Toxicity to aquatic algae and cyanobacteria.001 to 002

Toxicity to aquatic algae and cyanobacteria.001

Toxicity to aquatic algae and cyanobacteria.001

Administrative data

Purpose flag:

weight of evidence

Study result type:

experimental result

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report Date:

2006

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

other guideline: EPA-600-R-95-136

GLP compliance:

no

Test materials

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

yes

Test material identity

Identifier:

CAS number

Identity:

10049-04-4

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: powder
- Physical state: solid dissolved in deionized water
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Stability under test conditions: no data
- Storage condition of test material: no data
- Other: none

Details on properties of test surrogate or analogue material:

no data

Analytical monitoring:

no

Details on sampling:

- Concentrations: 0, 0.036, 0.071, 0.143, 0.285, 0.570 mg/L
- Sampling method: test concentrations were verified at the beginning of the test using HACH DPD Method 10126
- Sample storage conditions before analysis: no data

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was made from powered product supplied by Ecochlor. Two separate powders were mixed according to directions by dissolving them in deionized water and bringing the volume to one liter to obtain a target concentration of 3000 ppm chlorine dioxide.
- Eluate: deionized water
- Differential loading: 0.5 X dilution series
- Controls: only seawater
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data

Test organisms

Test organisms (species):

other: Macrocystis pyrifera

Details on test organisms:

TEST ORGANISM
- Common name: Macrocystis pyrifera
- Strain: no data
- Source: La Jolla Cove, San Diego
- Age at study initiation (mean and range, SD): Spores
- Length at study initiation (length definition, mean, range and SD): no data
- Weight at study initiation (mean and range, SD): no data
- Method of breeding: no data
- Feeding during test
- Food type: no data
- Amount: no data
- Frequency: no data

ACCLIMATION
- Acclimation period: no data
- Acclimation conditions (same as test or not): no data
- Type and amount of food: no data
- Feeding frequency: no data
- Health during acclimation (any mortality observed): no data

QUARANTINE (wild caught)
- Duration: no data
- Health/mortality: no data

Study design

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no data

Test conditions

Hardness:

no data

Test temperature:

15+-1°C

pH:

no data

Dissolved oxygen:

no data

Salinity:

32+-2 ppt

Nominal and measured concentrations:

- nominal conc.: 0, 0.036, 0.071, 0.143, 0.285, 0.570 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Petri dish
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 30 mL
- Aeration: none
- 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: not applicable
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Seawater from Scripps Institute, San Diego
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: no data
- Intervals of water quality measurement: no data

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 0, 0.036, 0.071, 0.143, 0.285, 0.570 mg/L 
- Results used to determine the conditions for the definitive study: no data

Reference substance (positive control):

yes

Remarks:

Copper chloride

Results and discussion

Effect concentrationsopen allclose all

Effect concentrations 1

Duration:

48 h

Endpoint:

EC50

Effect conc.:

ca. 0.202 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: germination

Remarks (e.g. 95% CL):

0.196-0.208

Effect concentrations 2

Duration:

48 h

Endpoint:

NOEC

Effect conc.:

ca. 0.143 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Germination and germ tube length

Details on results:

no data

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

Statistical analyses were calculated based on the measured value.

Any other information on results incl. tables:

Table: Results (means +- standard deviation) of the 48h toxicity test using M. pyrifera.

Chlorine dioxide (mg/L)			Germination (%)	Germ tube length (mg)
Nominal	Measured
0	-		94.0 ± 1.2	13.8 ± 1.1
0.036	-		93.2 ± 2.2	13.7 ± 0.8
0.071	-		93.8 ± 3.3	13.3 ± 0.6
0.143	-		97.2 ± 1.6	12.5 ± 1.3
0.285	-		0	0
0.57	-		0	0

Applicant's summary and conclusion

Conclusions:

Toxicity of the substance has been tested on M. pyrifera, in a 48h acute test. The results demonstrated a NOEC of 0.143 mg/L of chlorine dioxide for germination and germ tube length criteria; an EC50(48h) = 0.202 mg/L for germination, and an EC50(48h) = 0.206 mg/L for germ tube length criterion.

Executive summary:

Toxicity of the substance has been tested on Macrocystis pyrifera, in a 48h acute-static test, following US EPA Guideline EPA-600 -R-95 -136. A control and five different concentrations (0.036, 0.071, 0.143, 0.285, and 0.570 mg/L) were tested in 5 replicates. The results demonstrated a NOEC of 0.143 mg/L of chlorine dioxide for germination and germ tube length criteria; an EC50(48h) = 0.202 mg/L for germination, and an EC50(48h) = 0.206 mg/L for germ tube length criterion.

Validity criteria fulfilled:

no

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

Toxicity to aquatic algae and cyanobacteria.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:

No GLP and no standard test protocol, but test described in
enough detail.

Data source

Reference

Reference Type:

publication

Title:

Toxicity of chlorate to marine microalgae.

Author:

Stauber, J.L.

Year:

1998

Bibliographic source:

Aquatic toxicology 41 pp. 213-227

Materials and methods

Test guideline

Guideline:

other guideline: Standard 72h growth inhibition bioassay (Stauber etal., 1994)

Principles of method if other than guideline:

Toxicity of chlorate at 3 nitrate concentrations (<0.005, 1 and 15 mg/l NO3) was studies using a standard 72 hours growth inhibtion bioassay (Stauber et al., 1994). At a nitrate concentration of <0.005 mg/l the toxicity of chlorate at 5 concentrations (0.5, 1, 5, 10 and 20 mg/l) was determined. At higher nitrate concentration (1 and 15 mg/l), 5 concentrations of chlorate were tested ranging from 1-1000 mg/l depending on the alga and nitrate concentration in each experiment.
Cell densities were determined daily using a Coulter Multisizer IIE with 70 µm aperture.

GLP compliance:

no

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

Test material identity 2

Details on test material:

Test substance: other TS: Potassium chlorate (AnalaR)

Analytical monitoring:

yes

Details on sampling:

See details in analytical methods.

Vehicle:

no

Details on test solutions:

Three nitrate levels were used: <0.005, 1 and 15 mg/l NO3. At nitrate concentrations of <0.005 mg/l the toxicity of chlorate was tested at 0.5, 1, 5, 10 and 20 mg/l ClO3-. At nitrate concentrations of 1 and 15 mg/l the test concentrations were ranging from 1 to 1000 mg/l ClO3-, depending on the alga and nitrate concentration in each experiment.

Test organisms

Test organisms (species):

other: other algae: Nitzschia closterium

Details on test organisms:

Nitzschia closterium (Ehrenberg) W. Smith, originally isolated from Port Hacking, NSW, Strain CS-5c.
Culture medium: Medium f (Guillard and Ryther, 1962) with the iron and trace element concentrations halved.

Study design

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Nominal and measured concentrations:

At nitrate concentrations of <0.005 mg/l the toxicity of chlorate was tested at 0.5, 1, 5, 10 and 20 mg/l ClO3-. 
At nitrate concentrations of 1 and 15 mg/l the test concentrations were ranging from 1 to 1000 mg/l ClO3-, depending on the alga and nitrate concentration in each experiment.

Chlorate was measured in each bioassay flask at the beginning and end of the test and was always within 10% of the nominal chlorate concentration.

Details on test conditions:

Seawater for the preparation of the test medium was collected 0-1 km offshore from Port Hacking, NSW. The seawater was filtered through a 0.45 µm membrane filter and stored at 4 °C.

Reference substance (positive control):

no

Results and discussion

Effect concentrationsopen allclose all

Effect concentrations 1

Duration:

72 h

Endpoint:

EC50

Effect conc.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

other: chlorate ion

Basis for effect:

growth rate

Remarks (e.g. 95% CL):

95% CL: 1.6 - 2.3 mg/l Nitrate conc: < 0.005 mg/l

Effect concentrations 2

Duration:

72 h

Endpoint:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: chlorate ion

Basis for effect:

growth rate

Remarks (e.g. 95% CL):

Nitrate conc: 1 mg/l

Effect concentrations 3

Duration:

72 h

Endpoint:

EC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

other: chlorate ion

Basis for effect:

growth rate

Remarks (e.g. 95% CL):

Nitrate conc: 15 mg/l

Effect concentrations 4

Duration:

72 h

Endpoint:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: chlorate ion

Basis for effect:

growth rate

Remarks (e.g. 95% CL):

Nitrate conc: 15 mg/l

Details on results:

- Analytical results: Chlorate was measured in each bioassay flask at the beginning and end of the test and was always within 10% of the nominal chlorate concentration. Chlorate concentrations remained stable over the 3 days and chlorate was not reduced to chlorite or chloride in the light either in the presence or absence of algal cells.

Reported statistics and error estimates:

Data analyses were performed using Toxcalc version 5.0 (Tidepool scientific software).

Any other information on results incl. tables:

 Recalculation of the results from mg/l ClO3- to KClO3. EC50:
- <0.005 mg nitrate/l: 1.9 mg ClO3-/l (95% c.i. 1.6-2.3 mg/l) corresponds to 2.8 mg KClO3/l (95% c.i. 2.3-3.4 mg/l)
- 1 mg nitrate/l: 10 mg ClO3-/l corresponds to 15 mg KClO3/l 
- 15 mg nitrate/l: >500 mg ClO3-/l corresponds to >735 mg KClO3/l 
NOEC:- 15 mg nitrate/l: 100 mg ClO3-/l corresponds to 147 mg KClO3/l

Control cell division rate (doublings/day)
- <0.005 mg nitrate/l: 0.8
- 1 mg nitrate/l: 1.1
- 15 mg nitrate/l: 1.3

Remark from reviewer: According to OECD guideline 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test the 

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period. This corresponds to a specific growth rate of 0.92 day-1. For the test performed at <0.005 mg nitrate/l this was not the case and therefore this test result is not valid and can not be used. 

Applicant's summary and conclusion

Conclusions:

EC50: 
- 1 mg nitrate/l: 10 mg ClO3-/l corresponds to 15 mg KClO3/l
- 15 mg nitrate/l: >500 mg ClO3-/l corresponds to >734 mg KClO3/l

NOEC:- 15 mg nitrate/l: 100 mg ClO3-/l corresponds to 147 mg KClO3/l

The results obtained at a nitrate concentration of <0.005 mg/l were not considered to be valid due to a low doubling rate in the control vessels and can not be used, therefore.

Executive summary:

Toxicity of chlorate at 3 nitrate concentrations (<0.005, 1 and 15 mg/l NO3) was studies using a standard 72 hours growth inhibtion bioassay (Stauber et al., 1994). At a nitrate concentration of <0.005 mg/l the toxicity of chlorate at 5 concentrations (0.5, 1, 5, 10 and 20 mg/l) was determined. At higher nitrate concentration (1 and 15 mg/l), 5 concentrations of chlorate were tested ranging from 1-1000 mg/l depending on the alga and nitrate concentration in each experiment.

Cell densities were determined daily using a Coulter Multisizer IIE with 70 µm aperture.

Nitzschia closterium was used as test organism.

EC50:

- <0.005 mg nitrate/l: 1.9 mg ClO3-/l (95% c.i. 1.6-2.3 mg/l) corresponds to 2.8 mg KClO3/l (95% c.i. 2.3-3.4 mg/l)

- 1 mg nitrate/l: 10 mg ClO3-/l corresponds to 15 mg KClO3/l

- 15 mg nitrate/l: >500 mg ClO3-/l corresponds to >734 mg KClO3/l

NOEC:- 15 mg nitrate/l: 100 mg ClO3-/l corresponds to 147 mg KClO3/l

The results obtained at a nitrate concentration of <0.005 mg/l were not considered to be valid due to a low doubling rate in the control vessels and can not be used, therefore.