Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Bu and iso-Pr) esters, zinc salts

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols and standard test guidelines; however analysis of test material was not conducted except for TOC analysis

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The slope of the 96 hour dose response curve and the EC10 and EC90 values could not be calculated. These deviations had no effect on the outcome of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)

Deviations:

yes

Remarks:

The slope of the 96 hour dose response curve and the EC10 and EC90 values could not be calculated. These deviations had no effect on the outcome of the study

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Pr) esters, zinc salts

EC Number:

272-723-1

EC Name:

Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Pr) esters, zinc salts

Cas Number:

68909-93-3

Molecular formula:

Too complex

IUPAC Name:

Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Pr) esters, zinc salts

Details on test material:

- Name of test material (as cited in study report): CMA 601

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling and Recording of Cell Densities
The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscope examination with a haemocytometer. Cell counts were made and recorded daily during the 96 hour test.

Concentrations. A range finding test was conducted with a control and the WAF of 3 concentrations of test material 100, 300 and 1,000 mg/L. A second range finding test was conducted with a control and the WAF of 3 concentrations of test material at 1.0, 10, and 100 mg/L. A definitive test was conducted with five WAFs at 0.13, 0.5, 1.0, 5.0 and 10 mg/L.

Sampling for Analysis of Test Concentrations
TOC The measured concentrations of total organic carbon were determined in samples of test media collected at the beginning and end of the toxicity test. Samples were collected from each of the two replicate control and treatment vessels. Samples were passed through a 0.45 micron filter prior to analysis. They were stored in 125 mL amber glass bottles, preserved with sulfuric acid, and placed in a refrigerator. The TOC analysis was conducted according to US EPA Method 415.1, 40 CFR part 136).

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Each of the five WAFs was prepared by combining the appropriate amount of test substance and dilution water in a glass mixing vessel equipped with a magnetic stirrer and stirring these mixtures for approximately 24 hours, settling the mixtures for approximately 1 hour, and siphoning the water phase containing the WAF. During the mixing of the WAF the vortex extended approximately 50% of the distance from the surface to the bottom of the mixing vessel.

- Eluate: NA.
- Differential loading: NA.
- Controls: Negative controls
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): NA
- Concentration of vehicle in test medium (stock solution and final test solution): NA
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No insoluble material was noted in any test vessel containing test material

Test organisms

Test organisms (species):

other: Selenastrum capricornutum UTEX 1648

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain: Selenastrum capricornutum UTEX 1648
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Texas at Austin
- Age of inoculum (at test initiation): Culture was procured and delivered to T.R. Wilbury on February 24, 1994.
- Method of cultivation: The culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test. The inoculum used for testing had been actively growing for 7 days.

ACCLIMATION
- Acclimation period: At least 14 days.
- Culturing media and conditions (same as test or not): Water used for acclimation of test organisms and for all toxicity testing was sterile enriched media (US EPA 1978) adjusted to a pH of 7.5. The media has less than 1 mg/L total organic carbon and less than 10 mg/L total suspended solids at the beginning of the test and <5 mg/L total organic carbon and 30 mg/L total suspended solids at the end of the test (in part due to the presence of algal cells)

- Any deformed or abnormal cells observed: The number of algae cells/mL in each test vessel and the occurrence of relative size difference, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

At the conclusion of the test a subsample of test media from each flask containing the WAF and the 5.0 mg/L solution of test substance was combined with fresh media to determine whether algicidal or algistatic effects had occurred. This flask was incubated under test conditions for 6 days and examined for the presence of algal cells.

Test conditions

Hardness:

No data available from study report

Test temperature:

The temperature of incubator for the 96 hour duration ranged from 23.5 to 23.7 degrees C. The test was conducted at a target temperature of 22 to 25 degrees C with five concentrations of WAF and a dilution water control.

pH:

Initial pH values for controls and test solutions ranged from 7.3 to 7.7
Final pH values for controls and test solutions ranged from 7.4 to 10.5

Dissolved oxygen:

No data available from study report

Salinity:

Not applicable/freshwater

Nominal and measured concentrations:

Nominal loading rates were 0, 0.13, 0.5, 1.0, 5.0, and 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: The test was performed in 250 mL glass Erlenmeyer flasks that contained 100 mL of test solution
- Material, size, headspace, fill volume: 100 ml of test solution
- Aeration: Test vessels were randomly arranged on a rotary shaker adjusted to 100 rpm in an incubator during the test
- Type of flow-through (e.g. peristaltic or proportional diluter): NA
- Renewal rate of test solution (frequency/flow rate): NA
- Initial cells density: 1.00E+04 cells per ml.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS:
- Source/preparation of dilution water: not reported

OTHER TEST CONDITIONS
- Sterile test conditions:
- Adjustment of pH:
- Photoperiod: 24 hour light and 0 hour dark photoperiod was automatically maintained with cool white fluorescent lights that provided a light intensity of approximately 50 uEin/m2sec
- Light intensity and quality: 24 hour light and 0 hour dark photoperiod was automatically maintained with cool white fluorescent lights that provided a light intensity of approximately 50 uEin/m2sec

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 1.00E+04 cells per ml via haemocytometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: NA
- Range finding study
Two range finding tests were conducted. The first range finding test was conducted with a control and the WAF of 3 concentrations of test material, at 100, 300, and 1,000 mg/L. After 96 hours the number of algal cells/mL in flasks containing the 3 WAFs were less than 1% of the number of cells/mL in the control flasks. A second range finding test was conducted with a control and the WAF of 3 concentrations of test material at 1.0, 10, and 100 mg/L. After 96 hours the number of algal cells/mL in flasks containing the 100 and 10 mg/L WAF were less than 1 % of the number of cells in the control flasks and the number of cells/mL at 1.0 mg/L WAF were >100% of the control.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 4

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): The algal population grew well during the test, resulting in an average of 1,791,000 cells/mL in the control, resulting in a doubling time of 12.8 hours.

- Observation of abnormalities (for algal test):
- Unusual cell shape: There were no abnormalities detected in any of the control or test cultures
- Colour differences: The report states no effects on color was observed
- Flocculation: The report states no effects on flocculation was observed
- Adherence to test vessels: The report states no effects on adherence to test containers was observed
- Aggregation of algal cells: The report states no effects on aggregation of cells was observed

-An aliquot of test media taken from the 5.0 mg/L WAF at 96 hours when cultured in fresh media for an additional 144 hours, produced 284,000 cells/mL. The WAF of the test substance at 5.0 mg/L was therefore algistatic rather than algicidal

Results with reference substance (positive control):

No positive control data reported

Any other information on results incl. tables

Table 1.  Average specific growth rate and percent change from the control from the acute toxicity test with the WAF of

5 concentrations of test material and the freshwater alga, Selenastrum capricornutum

 

Nominal Concentration of WAF (mg/L)	Average Specific Growth Rate				Percent Change from Control
	24 hr	48 hr	72 hr	96 hr	24 hr	48 hr	72 hr	96 hr
0, Control	0.061	0.053	0.058	0.054	--	--	--	--
0.13	0.061	0.052	0.058	0.054	0	2	0	0
0.50	0.053	0.052	0.058	0.054	13	2	0	0
1.0	0.052	0.050	0.057	0.053	15	6	2	2
5.0	0.00	0.000	0.000	0.000	100	100	100	100
10	0.00	0.002	0.000	0.000	100	96	100	100

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Selenastrum capricornutum to CMA#601 test material gave 72 hour and 96 hour EC50s of 2.0 mg/L based on the number of cells and 2.1 mg/L based on the average specific growth rate.

No effects (size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted.

Based on the number of cells/mL or the average specific growth rate, the 72 and 96 hour NOEC values are 1.0 mg/L.

An aliquot of test media taken from the 5.0 mg/L WAF at 96 hours when cultured in fresh media for an additional 144 hours, produced 284,000 cells/mL. The WAF of the test substance at 5.0 mg/L was therefore algistatic rather than algicidal.

Executive summary:

Introduction.A study was performed to assess the effect of the test material on the growth of the freshwater algaSelenastrum capricornutum. The method followed that described in the OECD Guidelines for Testing of Chemicals, guideline No. 201, “Algal Growth Inhibition Test”.

Methods. The test was performed under static conditions at 22 to 25 deg. C with the WAF of five concentrations of test material and a dilution water control. The dilution water was sterile synthetic media adjusted to a pH of 7.5. Nominal concentrations of WAF were 0, 0.13, 0.50, 1.0, 5.0, and 10 mg/L. The five WAF samples were formulated by combining the test substance and dilution water in a glass mixing vessel equipped with a magnetic stirrer, mixing the solution for approximately 24 hours, settling the solution for approximately 1 hour, and siphoning the water phase containing the water accommodated fraction. During the mixing of the WAF the vortex extended approximately 50% of the distance from the surface to the bottom of the mixing vessel. The test substance was not heated prior to formulation of the WAF samples, and no undissolved material was observed in the WAF solutions. Nominal concentrations of WAF were used for all calculations.

Results.Exposure ofSelenastrum capricornutumto the test material gave 72 hour and 96 hour EC50 of 2.0 mg/L based on (nominal loading) the number of cells and 2.1 mg/L based on the average specific growth rate.

No effects (size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted. 

Based on the number of cells/mL or the average specific growth rate, the 72 and 96 hour NOEC (nominal loading) values are 1.0 mg/L. 

An aliquot of test media taken from the 5.0 mg/L WAF at 96 hours when cultured in fresh media for an additional 144 hours, produced 284,000 cells/mL. The WAF of the test substance at 5.0 mg/L was therefore algistatic rather than algicidal.

The study met the acceptability criteria prescribed by the protocol and was considered valid.