Short-term toxicity to fish.002 to .003

Short-term toxicity to fish.002

Administrative data

Purpose flag:

key study

Study result type:

experimental result

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

No purity provided in the CoA and no batch number provided. The method would have been better run under flow-through conditions considering the potential for loss of chlorine dioxide due to its volatility.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Remarks:

but no certificate is joined

Test materials

Identity of test material same as for substance defined in section 1 (if not read-across):

yes

Test material identityopen allclose all

Test material identity 1

Identifier:

CAS number

Identity:

10049-04-4

Test material identity 2

Identifier:

EC number

Identity:

233-162-8

Details on test material:

- Name of test material (as cited in study report): Chlorine dioxide
- Molecular formula (if other than submission substance): no data
- Molecular weight (if other than submission substance): no data
- Smiles notation (if other than submission substance): no data
- InChl (if other than submission substance): no data
- Structural formula attached as image file (if other than submission substance): no data
- Substance type: Yellow liquid
- Physical state: liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: 2 g/L i.e. 0.2 % of chlorine dioxide (nominal)
- Isomers composition: no data
- Purity test date: prepared on 1995-03-09
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: 6 months from the preparation date
- Storage condition of test material: in the dark at +4°C

Details on properties of test surrogate or analogue material:

no data

Analytical monitoring:

yes

Details on sampling:

Oxygen, pH, temperature and the test substance concentration were measured daily.

- Concentrations: 
- nominal concentrations: 0.009, 0.021, 0.045, and 0.1 mg/L
- measured concentrations: 0.01, 0.02, 0.05, 0.08 mg/L
- Sampling method: samples were analyzed for chlorine dioxide by RBM, with the spectrophotometer HACH and the method supplied by the Sponsor.
- Sample storage conditions before analysis: no data

Vehicle:

no

Details on test solutions:

Some volatility of chlorine dioxide may be expected. Although the test systems were not treated as such, analysis of the fresh test solutions showed that there was low loss during preparation.

Test organisms

Test organisms (species):

Brachydanio rerio (new name: Danio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: no data
- Source: AQUA, Turin, IT
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): 3 cm
- Weight at study initiation (mean and range, SD): 0.4 g
- Method of breeding: no data
- Feeding during test: NO
- Food type:
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period: 2 wks before start of test. All fish were exposed (in a glass aquarium) to water.
- Acclimation conditions (same as test or not): in water , from municipal water main system, at 22°C, pH: 7.2, Hardeness: 21 mg/L of CaCO3, [O2]: 8.1 mg/L
- Type and amount of food: Mangine SERA (Aquaristik GmbH, batch CH 09-3)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no mortality was recorded after 7 days of acclimation

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

no data

Test conditions

Hardness:

no data

Test temperature:

21 - 22 °C

pH:

6.6 - 7.1

Dissolved oxygen:

7.7 - 9.0

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 0.009, 0.021, 0.045 and 0.1 mg/L

Measured concentrations at t = 0 h: 0.01, 0.02, 0.05 and 0.08 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 5 L
- Aeration:no data
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- Renewal rate of test solution (frequency/flow rate): no data
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: no data

OTHER TEST CONDITIONS
- Adjustment of pH: 
- Photoperiod: 16:8
- Light intensity: no data

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 
- nominal concentrations: 0.009, 0.021, 0.045, and 0.01 mg/L
- measured concentrations: 0.01, 0.02, 0.05, and 0.08 mg/L

Reference substance (positive control):

no

Results and discussion

Effect concentrationsopen allclose all

Effect concentrations 1

Duration:

48 h

Endpoint:

LC50

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

mortality

Remarks (e.g. 95% CL):

95 % confidence limits 0.04 – 0.061 mg/L

Effect concentrations 2

Duration:

72 h

Endpoint:

LC50

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

mortality

Remarks (e.g. 95% CL):

95 % confidence limits 0.04 – 0.061 mg/L

Effect concentrations 3

Duration:

96 h

Endpoint:

LC50

Effect conc.:

0.021 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

mortality

Remarks (e.g. 95% CL):

95 % confidence limits 0.01 – 0.04 mg/L

Effect concentrations 4

Duration:

96 h

Endpoint:

LC0

Effect conc.:

0.009 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Effect concentrations 5

Duration:

96 h

Endpoint:

LC100

Effect conc.:

0.072 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

mortality

Details on results:

The concentration causing no mortality was lower than 0.009 mg/L. The lowest concentration causing 100% mortality was 0.100 mg/L. In the fish treated at 0.045 and 1.00 mg/L the only sign of abnormality observed was decreased escape reflex that appeared at the 24 h observation until the 72 observation in the fish (in fish treated with 0.045 mg/L. In animals treated at 0.009 and 0.021 mg/L the only sign of abnormality observed was decreased escape reflex that appeared at the 72 h observation in those treated at 0.009 mg/L. In both the treated groups this sign lasted until the end of the study. The study was performed taking into account the actual content of the stock solution of chlorine dioxide. The toxicity values can therefore be expressed as referring to the 100% ClO2 and to the solution at the nominal concentration of 0.2%. The LC50 at 24 h was not calculable.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

no data

Any other information on results incl. tables:

Table A7_4_1_1(1)-5:        Mortality data

Test-Substance Concentration nominal  [mg/l]	Mortality
	Number     24 h          48 h          72 h          96 h				Percentage     24 h         48 h          72 h           96 h
0.0	0/7	0/7	0/7	0/7	0	0	0	0
0.009	0/7	0/7	0/7	1/7	0	0	0	14
0.021	0/7	0/7	0/7	3/7	0	0	0	43
0.045	0/7	1/7	1/7	4/7	0	14	14	57
0.100	4/7	7/7	7/7	7/7	57	100	100	100

Table A7_4_1_1(1)-6:        Effect data

	48 h [mg/l]1	95 % c.l.	96 h [mg/l]1	95 % c.l.
LC0	Not stated	Not stated	0.009 (n)	Not stated
LC50	0.054 (n)	Not stated	0.030 (n)	Not stated
LC100	Not stated	Not stated	0.100 (n)	Not stated

1 effect data are based on nominal (n) concentrations

Applicant's summary and conclusion

Conclusions:

96 h LC50 = 0.021 mg/L (based on the TWA)

Executive summary:

Acute toxicity test was conducted on zebrafsih, Danio rerio, to assess effects of chlorine dioxide. The experiment was realized under GLP conditions, and following C.1 EEC Guideline. 4 measured concentrations were tested, with 7 fish exposed to each ones: 0.01, 0.02, 0.05, and 0.08 mg/L of chlorine dioxide. LC50(96h) = 0.030 mg/L of 100 % ClO2; i.e. 0.015 g/L of 0.2% ClO2

Validity criteria fulfilled:

no

Remarks:

no reference substance was tested

Administrative data

Purpose flag:

supporting study

Study result type:

experimental result

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

No guideline or GLP compliance stated

Data source

Reference

Reference Type:

publication

Title:

Comparison of chlorine and chlorine dioxide toxicity to fathead minnows and bluegill.

Author:

Wilde EW, Soracco JR Mayack LA, Shealy RL, Broadwell TL and Steffen RF

Year:

1983

Bibliographic source:

Water Resources, 17(10): 1327-1331

Materials and methods

Principles of method if other than guideline:

Flow-through 96 hour bioassays were conducted on juvenile and adult fathead minnows using a mobile laboratory located adjacent to Par Pond, to test ClO2 toxicity. Dissolved oxygen, temperature, conductivity and pH in the test chambers were measured in selected chambers.

GLP compliance:

no data

Test materials

Identity of test material same as for substance defined in section 1 (if not read-across):

yes

Test material identityopen allclose all

Test material identity 1

Test material identity 2

Analytical monitoring:

yes

Details on sampling:

Measurements of dissolved oxygen, temperature, conductivity and pH in the test chambers receiving 100, 32, 5.6 and 0% doses of the b iocide stock solutions were made at the beginning of the tests and at 24 hour intervals during the tests (Hydrolab 8000 was used).

Details on test solutions:

ClO2 solutions were prepared by adding previously standardised mixture of NaOCl, HCl and sodium chlorite. Stock solutions were added to their respective dilutor systems for 1 hour at approximately 0, 24, 48 and 72 hours into the testing period. Two stock solutions were prepared 30 minutes apart, 15 minutes prior to their use during thedosing period. One was used during the first half hour and the other during the remainder of the dosing period, a procedure used to compensate for the degradation of the solutions. 100, 56, 32,18, 5.6 and 0% of stock solution of the biocide was delivered to duplicate test chambers using Par Pond as a dilutent. The dilutor systems were adjusted to cycle every 1.8 min resulting in 22.9 volume changes per day in each chamber. However, during the daily biocide exposure periods, and one hour thereafter, systems were adjusted to a cycle time of 1.5 in to achieve one complete volume change an hour.

Test organisms

Test organisms (species):

Pimephales promelas

Details on test organisms:

* Juvenile fathead minnows - age: 4 weeks old; feed: brine shrimp (Artemia sp.) daily during acclimation and test periods to prevent starvation
* Adult fathead minnows - age: 1 year old; feed: not fed

Fish were acclimated to Par Pond water for 3 days prior to the initiation of the bioassays. During the first 24 h the fish were slowlly acclimated from well water (approx. 22oC, pH8.2) to Par Pond water (approx 28oC, pH 7.0).

Study design

Test type:

flow-through

Water media type:

freshwater

Total exposure duration:

96 h

Test conditions

Test temperature:

Mean ± S.D.: 27.7 ± 0.2oC
Range: 24.5 - 30.4oC

pH:

Mean ± S.D.: 7.0 ± 0.1
Range: 6.7 - 7.5

Dissolved oxygen:

Mean ± S.D.: 6.8 ± 0.1 mg/L
Range: 3.8 - 7.9 mg/L

Details on test conditions:

CONDUCTIVITY:
Mean ± S.D.: 69.9 ± 0.3 µmhos/cm
Range: 64 - 81 µmhos/cm

Results and discussion

Effect concentrationsopen allclose all

Effect concentrations 1

Effect concentrations 2

Details on results:

In mg/L:
* Juvenile fathead minnows
96-h peak: 0.07 (0.06 - 0.08)
96-h mean max: 0.06 (0.05 - 0.07)
96-h mean: 0.02 (0.01 - 0.02)
96-h accum.: 3.26 (2.10 - 4.54)
(Chi-square is small: P > 0.10)

* Adult fathead minnows
96-h peak: 0.63 (0.33 - 2.74)
96-h mean max: 0.46 (0.25 - 1.61)
96-h mean: 0.17 (0.13 - 0.24)
96-h accum.: 93.47 (40.97 - 327.83)

Reported statistics and error estimates:

Median lethal concentrations (96-h LC50s) were determined by probit analysis. Log transformations of dose values were not used , because a bettter goodness of fit was obtained in the majority of cases by using actual dose values. Computations were performed using an IBM computer and a Statistical Analysis System (SAS) program. Biocide doses were calculated as follows:
(1) 96-h peak = the single highest biocide residual level detected during the tests
(2) 96-h mean maximum = the average maximum biocide residual detected during the 4 days of testing
(3) 96-h mean = the mean biocide residual level during the four approx. 2 h exposure periods.
(4) 96-h accumulative exposure = the total 96-h biocide exposure in mg/L residual x min of exposure (area under a time-concnetration curve).

Applicant's summary and conclusion

Short-term toxicity to fish.003

Administrative data

Purpose flag:

key study

Study result type:

experimental result

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

No purity provided in the CoA and no batch number provided. The method would have been better run under flow-through conditions considering the potential for loss of chlorine dioxide due to its volatility.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Remarks:

but no certificate is joined

Test materials

Identity of test material same as for substance defined in section 1 (if not read-across):

yes

Test material identityopen allclose all

Test material identity 1

Identifier:

CAS number

Identity:

10049-04-4

Test material identity 2

Identifier:

EC number

Identity:

233-162-8

Details on test material:

- Name of test material (as cited in study report): Chlorine dioxide
- Molecular formula (if other than submission substance): no data
- Molecular weight (if other than submission substance): no data
- Smiles notation (if other than submission substance): no data
- InChl (if other than submission substance): no data
- Structural formula attached as image file (if other than submission substance): no data
- Substance type: Yellow liquid
- Physical state: liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: 2 g/L i.e. 0.2 % of chlorine dioxide (nominal)
- Isomers composition: no data
- Purity test date: prepared on 1995-03-09
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: 6 months from the preparation date
- Storage condition of test material: in the dark at +4°C

Details on properties of test surrogate or analogue material:

no data

Analytical monitoring:

yes

Details on sampling:

Oxygen, pH, temperature and the test substance concentration were measured daily.

- Concentrations: 
- nominal concentrations: 0.009, 0.021, 0.045, and 0.1 mg/L
- measured concentrations: 0.01, 0.02, 0.05, 0.08 mg/L
- Sampling method: samples were analyzed for chlorine dioxide by RBM, with the spectrophotometer HACH and the method supplied by the Sponsor.
- Sample storage conditions before analysis: no data

Vehicle:

no

Details on test solutions:

Some volatility of chlorine dioxide may be expected. Although the test systems were not treated as such, analysis of the fresh test solutions showed that there was low loss during preparation.

Test organisms

Test organisms (species):

Brachydanio rerio (new name: Danio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: no data
- Source: AQUA, Turin, IT
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): 3 cm
- Weight at study initiation (mean and range, SD): 0.4 g
- Method of breeding: no data
- Feeding during test: NO
- Food type:
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period: 2 wks before start of test. All fish were exposed (in a glass aquarium) to water.
- Acclimation conditions (same as test or not): in water , from municipal water main system, at 22°C, pH: 7.2, Hardeness: 21 mg/L of CaCO3, [O2]: 8.1 mg/L
- Type and amount of food: Mangine SERA (Aquaristik GmbH, batch CH 09-3)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no mortality was recorded after 7 days of acclimation

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

no data

Test conditions

Hardness:

no data

Test temperature:

21 - 22 °C

pH:

6.6 - 7.1

Dissolved oxygen:

7.7 - 9.0

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 0.009, 0.021, 0.045 and 0.1 mg/L

Measured concentrations at t = 0 h: 0.01, 0.02, 0.05 and 0.08 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 5 L
- Aeration:no data
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- Renewal rate of test solution (frequency/flow rate): no data
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: no data

OTHER TEST CONDITIONS
- Adjustment of pH: 
- Photoperiod: 16:8
- Light intensity: no data

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 
- nominal concentrations: 0.009, 0.021, 0.045, and 0.01 mg/L
- measured concentrations: 0.01, 0.02, 0.05, and 0.08 mg/L

Reference substance (positive control):

no

Results and discussion

Effect concentrationsopen allclose all

Effect concentrations 1

Duration:

48 h

Endpoint:

LC50

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

mortality

Remarks (e.g. 95% CL):

95 % confidence limits 0.04 – 0.061 mg/L

Effect concentrations 2

Duration:

72 h

Endpoint:

LC50

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

mortality

Remarks (e.g. 95% CL):

95 % confidence limits 0.04 – 0.061 mg/L

Effect concentrations 3

Duration:

96 h

Endpoint:

LC50

Effect conc.:

0.021 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

mortality

Remarks (e.g. 95% CL):

95 % confidence limits 0.01 – 0.04 mg/L

Effect concentrations 4

Duration:

96 h

Endpoint:

LC0

Effect conc.:

0.009 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Effect concentrations 5

Duration:

96 h

Endpoint:

LC100

Effect conc.:

0.072 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

mortality

Details on results:

The concentration causing no mortality was lower than 0.009 mg/L. The lowest concentration causing 100% mortality was 0.100 mg/L. In the fish treated at 0.045 and 1.00 mg/L the only sign of abnormality observed was decreased escape reflex that appeared at the 24 h observation until the 72 observation in the fish (in fish treated with 0.045 mg/L. In animals treated at 0.009 and 0.021 mg/L the only sign of abnormality observed was decreased escape reflex that appeared at the 72 h observation in those treated at 0.009 mg/L. In both the treated groups this sign lasted until the end of the study. The study was performed taking into account the actual content of the stock solution of chlorine dioxide. The toxicity values can therefore be expressed as referring to the 100% ClO2 and to the solution at the nominal concentration of 0.2%. The LC50 at 24 h was not calculable.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

no data

Any other information on results incl. tables:

Table A7_4_1_1(1)-5:        Mortality data

Test-Substance Concentration nominal  [mg/l]	Mortality
	Number     24 h          48 h          72 h          96 h				Percentage     24 h         48 h          72 h           96 h
0.0	0/7	0/7	0/7	0/7	0	0	0	0
0.009	0/7	0/7	0/7	1/7	0	0	0	14
0.021	0/7	0/7	0/7	3/7	0	0	0	43
0.045	0/7	1/7	1/7	4/7	0	14	14	57
0.100	4/7	7/7	7/7	7/7	57	100	100	100

Table A7_4_1_1(1)-6:        Effect data

	48 h [mg/l]1	95 % c.l.	96 h [mg/l]1	95 % c.l.
LC0	Not stated	Not stated	0.009 (n)	Not stated
LC50	0.054 (n)	Not stated	0.030 (n)	Not stated
LC100	Not stated	Not stated	0.100 (n)	Not stated

1 effect data are based on nominal (n) concentrations

Applicant's summary and conclusion

Conclusions:

96 h LC50 = 0.021 mg/L (based on the TWA)

Executive summary:

Acute toxicity test was conducted on zebrafsih, Danio rerio, to assess effects of chlorine dioxide. The experiment was realized under GLP conditions, and following C.1 EEC Guideline. 4 measured concentrations were tested, with 7 fish exposed to each ones: 0.01, 0.02, 0.05, and 0.08 mg/L of chlorine dioxide. LC50(96h) = 0.030 mg/L of 100 % ClO2; i.e. 0.015 g/L of 0.2% ClO2

Validity criteria fulfilled:

no