Long-term toxicity to aquatic invertebrates.002 to 003

Long-term toxicity to aquatic invertebrates.002

Administrative data

Purpose flag:

key study

Study result type:

read-across from supporting substance (structural analogue or surrogate)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

Study to GLP with analysis and analysis certificate. 1xquality criteria not met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report Date:

2008

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Principles of method if other than guideline:

0.2mg/c not fed to the adult animals due to adverse effects on test substance detection.
Test was extended to day 22 due to laying activity in all reps at the end of day 21.
60 juveniles per survivng adult was not achieved.

GLP compliance:

yes (incl. certificate)

Test materials

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

no

Test material identityopen allclose all

Test material identity 1

Identifier:

CAS number

Identity:

7758-19-2

Test material identity 2

Identifier:

EC number

Identity:

231-836-6

Details on test material:

Name: Sodium chlorite
chemical name : Sodium chlorite
Purity / active ingredient: 80.6 %
CAS reg. no: 7758-19-2
appearance: White powder 
water solubility: 571-572 g/l at 20°C
vapour pressure: 1.1 x 10-7 Pa at 25 °C
Lot number: 1358025
Stability: Sample is stable in condition received. 
Stability in water Stable in water and under test conditions down to 1 mg/l but relatively unstable at concentrations below 100 µg/l.
Storage until required: At room temperature away from light

The test substance, project sample code T 080006 was supplied by Fluka chemicals. Data on the handling, stability, composition, purity or other 
characteristics of the test substance supplied was used without further verification.

Analytical monitoring:

yes

Details on sampling:

Initially samples of every test concentrations were taken once a week. But the test substance appeared to be less stable than initially anticipated most likely due to organic matter build up. Therefore to increase accuracy, sampling frequency was increased to at least two times a week. Samples were 
filtered over a Pall 0.45 μm GHP Acrodisc filter, transferred into 5 ml IC vials, and analysed immediately..

Vehicle:

no

Details on test solutions:

Stock Solutions
To prepare the stock solutions, 0.04 ± 0.0005 g of the test substance was weighed out, then dissolved directly into 10 litres (determined using a volumetric flask) of de-ionised water in 10 litre Duran glass bottles and mechanically agitated using a magnetic stirrer. Previous non-GLP studies on stability have revealed that the test substance is stable for more than one week in de-ionised water. The obtained preparations were agitated mechanically for between 30 minutes and 2 hours in an attempt to completely dissolve the test substance.
Test solutions
Test solutions were prepared by further dilution of the stock solution with M4 containing algae under flow through conditions. The stock was 
pumped at a known rate into the dilution water and allowed to mix directly in the inlet pipe shortly before reaching the test aquaria. This minimised 
contact of the test substance with the algae thereby optimising stability.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test animals were taken from a Daphnia magna clone 5 stock, (origin: Dr .U. Noack-Laboratorium Kathe-Paulus- Str. 1, D-31157 Sarstedt, 
Germany) The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and 
aging between 2-4 weeks (having previously produced at least one brood before use).

Test organisms were referenced tested prior to test with potassium dichromate as part of the laboratorium GLP maintainence.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

22 d

Remarks:

Extended by 1 day on request of SD.

Post exposure observation period:

No post exposure observation period. Adult daphnia examined during length measurment. No abnormalities observed.

Test conditions

Hardness:

Min- 10.0 dºH Max-15.2dºH

Test temperature:

Min- 21.3 Max-22.0°c

pH:

Min-6.9 Max-7.8

Dissolved oxygen:

Min- 7.1 Max-8.8 mg/l

Salinity:

Not measured

Nominal and measured concentrations:

Nominal= 12.5, 25.0, 50.0, 100, 200 µg/l
Measured concentrations were within +/- 20% of nominal concentrations calculated with time weighted average, with the exception of 1 replicate. 
Results in this replicate were compared for statistical significance and concluded a valid replicate. All calculations were therefore based on nominals.

Details on test conditions:

Test was conducted under flow through conditions with two replicates of 20 animals and a replacement rate of at least 10 turnovers per day.

Reference substance (positive control):

yes

Remarks:

Acute reference with test substance conducted and reference substance potassium dichromate.

Results and discussion

Effect concentrationsopen allclose all

Effect concentrations 1

Duration:

22 d

Endpoint:

NOEC

Effect conc.:

25 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: reproductive output, weight of adult daphnids and on parent body length.

Effect concentrations 2

Duration:

22 d

Endpoint:

EC50

Effect conc.:

87 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Parent Mortality

Remarks (e.g. 95% CL):

95%CL

Effect concentrations 3

Duration:

22 d

Endpoint:

EC10

Effect conc.:

55 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Adult Mortality

Remarks (e.g. 95% CL):

95% CL

Effect concentrations 4

Duration:

22 d

Endpoint:

NOEC

Effect conc.:

15.02 µg/L

Conc. based on:

other: calculated as chlorite concentration

Basis for effect:

other: reproductive output, weight of adult daphnids, parent body length

Details on results:

Reproductive output,weight of adult and parental bodylength all resulted in the same NOEC values after stastical analysis (25µg/l of test material)
Adult mortality endpoints were higher therfore the lowest endpoint calculated was taken as the NOEC. After correction for active ingredient this 
equated to 15.02µg/l of actual chlorite. (80.6% purity)

Results with reference substance (positive control):

Test animals were subjected to a reference test as part of standard laboratory maintainence / procedure. No further details included in report.

Reported statistics and error estimates:

All data was tested for normality using the Shapiro wilks test or the Kolmogorov D test..The Bartlett’s test or the F-Test were used to check fo
r homogeneity of variance. Analysis of variance was performed on the number of living neonates per replicate using the Bonferroni t-test and 
verified with a second multiple comparison method, the Dunnett’s test.The Bonferroni test found a statistical effect compared to the control (p>0.05) at 100 µg/L while the LOEC found in the Dunnett’s test was 50 µg/l. For this reason the lowest NOEC based on reproduction of 25 µg/L is retained.

Further statistical analyses were performed using length and weight data. Both length and weight data were found to be normally distributed and 
displayed homogeneity of variance when the two highest concentrations (which exhibited significant parent mortality were excluded. 
Multi-comparison tests of group weights and length of parent animals were employed. Significant differences were found at 50 µg/L for both length and dry weight. The lowest NOEC based on length and weight is therefore 25 µg/L.

Any other information on results incl. tables:

For tables please see Annex in attached report. Multiple results were taken for 4 different endpoints. All results cannot be tabulated here. Stability results in preliminary tests are also included in the attached report.

Quality criteria

•  The mortality in the controls (parent females) should not exceed 20% at the end of the test. The mortality was 20%

•   The validity criteria for the coefficient of variation (less than 25% in the control based on the number of living neonates for each replicate at the end of the test) was achieved.

• The reproduction of 60 juveniles per living adult at the beginning of the test was not met.

Applicant's summary and conclusion

Conclusions:

Due to this study not meeting the reproduction criteria in the control validity of this study must be carefully considered. A decision was made by the study director to intentionally keep algae concentration (food) low to allow accurate analysis and guarantee exposure. This choice was made as a 
result of previous stability tests of chlorite with algae. These tests also conducted under GLP demonstrated that virtually no recovery was achieved at chlorite concentrations of up to 19.5µg/l. Therefore a flow through method was chosen. Statistically all endpoints with the exception of adult 
mortality resulted in the same NOEC calculation. This gives good confidence of genuine effect. The lower concentration of algae used serves also to
further increases the sensitivity of the testing system giving an absolute worst case senario. This study can therefore be considered reliable with 
restrictions mentioned.

Executive summary:

The EC50 and EC10for parental mortality was found to be 87 and 55 µg/l, respectively. The final result for this study is therefore based on the reproductive output, weight of adult daphnids and on parent body length values of 25 µg/l. Actual chlorite concentration 15.02µgl

Validity criteria fulfilled:

yes

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assign

Long-term toxicity to aquatic invertebrates.003

Administrative data

Purpose flag:

key study

Study result type:

read-across from supporting substance (structural analogue or surrogate)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

Study generated according to a valid and
internationally accepted testing guideline and was performed
under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report Date:

2004

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Test materials

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

no

Test material identityopen allclose all

Test material identity 1

Identifier:

CAS number

Identity:

7775-09-9

Test material identity 2

Identifier:

EC number

Identity:

231-887-4

Test material identity 3

Identifier:

EC name

Identity:

sodium chlorate

Details on test material:

- Appearance: Yellow granules/crystals
- Stability: Assumed to be stable under test conditions
- Purity: 99.66%
- water solubility 715 g/l at 20°C
- vapour pressure: 3.5 x 10E-5 Pa at 25°C
- batch number: 1E0103 WF
- storage until required: at room temperature away from light

Analytical monitoring:

yes

Details on sampling:

- Samples were taken on the first day of the study on preparation and just before changing. 
- Further samples were taken weekly. 
- Samples were taken in duplicate and were frozen until analysis.

Vehicle:

no

Details on test solutions:

- Procedures: To prepare the stock solutions, approximately 5 or 10 g of test substance was weighed and dissolved directly in 500 or 1000 ml, 
respectively of M4 medium. 
- The obtained preparations were agitated mechanically for 24 h in an attempt to completely dissolve the test substance. 
- The pH of each stock solution was between 6.8 and 8.3. 
- The pH was adjusted to between 7.6 and 7.8 with 1 M HCI or NaOH (reagent grade) when necessary. 
- A fresh stock solution was prepared for each solution change.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna STRAUS-clone 4 
- Source/supplier: AquaSense, Amsterdam, The Netherlands 
- Breeding method: According to the relevant SOP 
- Age: less than 24 hours 
- Feeding: Chlorella vulgaris 
- Feeding during test: yes, between 0.1 and 0.2 mg of carbon per daphnid per day, from day 8

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

133-138 °dH

Test temperature:

20.2-21.4 °C

pH:

7.6-8.2

Dissolved oxygen:

8.0-9.1 mg/l

Nominal and measured concentrations:

- Nominal: 0, 12.8, 32, 80, 200 and 500 mg/L 
- Measured: The measured concentrations were within 20% of the nominal concentrations and therefore the nominals were used for all 
statistical evaluation.

Details on test conditions:

DILUTION WATER: 
- Elendt M4 medium 
- Aeration: yes 
- Ca/Mg ratio: 4:1 
- Na/K ratio: 10:1 
- pH: 8.0 ± 0.5 
- Holding water: same as dilution water
- Renewal of test solution: every two to three days 
- Exposure vessel type: 50 ml glass beakers 
- Number of replicates, individuals per replicate: 10, 1
- Conductance: in control 609-663 µs/cm, in 500 mg/L 1099-1145 µs/cm
- Adjustment of pH: no
- Intensity of irradiation: ambient light provided by fluorescent tubes 
- Photoperiod: 16 hours photoperiod daily

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

21 d

Endpoint:

NOEC

Effect conc.:

>= 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction, weight and length

Details on results:

- Unhatched eggs: None found 
- Length and weight of surviving animals: No signifacnt difference between the concentrations. 
- Numbers of dead young: At all concentrations except the control at least one and at most five neonates were found to be dead. 

The mortality appeared to be concentration related but was insignificant compared to the living neonates. Immobilization: one daphnid died in 80 mg/L on day 15. 
- EC50 for mortality of adults and for reproduction could not be determined due to insufficient mortality in the test concentrations. 

CONTROL:
- Number/percentage of animals showing adverse effects: No mortality occurred 
- Average number of juveniles per parent: 103.3 after 21 days

Reported statistics and error estimates:

Weight data were tested for normality using Chi-square test and Barlett's test for homogeneity of variance. 
ANOVA was performed on the number of living neonates per parent using Bonferroni t-test and verified with the Dunnett's test.
All other parameters were not tested because the result in the control group was lower than in the test concentrations.

Any other information on results incl. tables:

Time to first brood: Number of animals having the first brood on a given day.

Conc (mg/l)	Day of first brood
	8	9	10	11	12	13
0	7	3
12.8	9		1
32	6	3	1
80	6	3		1
200	9	1
500	3	2	4			1

Number of juveniles produced: No significant difference was found in total and mean number of juveniles between the concentrations.  

Average total number of living juveniles produced per parent per concentration:

Conc (mg/l)	no. juveniles/parent
0	103.3
12.8	109.8
32	120.7
80	121.7
200	126.5
500	108.6

Applicant's summary and conclusion

Conclusions:

Test conducted under GLP with analysis, analysis certificate was present and to relevant guideline was used. This study can be considered reliable 
without restrictions.
The NOEC for reproduction, weight and length is equal to or greater than 500 mg/l.

Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 4, in a 21-day semi-static test complying with the OECD Guideline No. 211 , 21st September 1998.

The test criterion of toxicity used was reproductive capacity expressed as the number of neonates per daphnid per day.

The nominal concentrations used in the study were as follows: 0, 12.8, 32.0, 80.0, 200 and 500 mg/l

Analytical determinations of the test solutions were made on six occasions during the test. The concentrations were found to remain stable to within ±20% of the nominals. The nominal concentrations were used to calculate the effect concentrations.

The validity criteria were respected: No mortality occurred in the control group over the test period.

The average number of juveniles per parent in the control was 103.3 after 21 days The No Observed Effect Concentration (NOEC) is determined as the concentration used in the study that is immediately below the Lowest Observed Effect Concentration (LOEC), the latter derived statistically from the data, where possible, using the appropriate statistical test.

Reproductive output and length of adults at the end of the study were lower in the control than in any other concentration tested and was therefore not evaluated statistically. Weight data were found to be normally distributed and homogeneous. Using Dunnett’s and Bonferroni-t tests, the lowest Observed Effect Concentration (LOEC) based on weight was found to be greater than 500 mg/l. Based on these statistical results the NOEC for reproduction, weight and length is 500 mg/l.

The EC50 for adult mortality and for reproduction could not be determined due to insufficient mortality in any of the test concentrations.

Validity criteria fulfilled:

yes

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