Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study; restriction due to test material not being measured analytically

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

the 100 mg/L test concentration was incorrectly prepared resulting in a 220 mg/L stock solution;e the test concentration ratio between the 32 and 220 mg/l test concentrations was 6.9; the test concentration ratio between the 220 and 320 mg/L was 1.5

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

the 100 mg/L test concentration was incorrectly prepared resulting in a 220 mg/L stock solution;e the test concentration ratio between the 32 and 220 mg/l test concentrations was 6.9; the test concentration ratio between the 220 and 320 mg/L was 1.5

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate)

EC Number:

224-235-5

EC Name:

Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate)

Cas Number:

4259-15-8

Molecular formula:

Too complex

IUPAC Name:

1-Hexanol, 2-ethyl-, O,O-diester with phosphorodithioic acid, zinc salt

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
1) OECD 2000. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
2) ECETOC 1996. Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic observations of the WAFs were performed after filtering and showed there to be no microdispersions of test material to be present.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: Scenedesmus subspicatus, strain CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP) Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria.

- Method of cultivation: Cultures were maintained in the laboratory at a temperature of 21 degrees C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Temperature was maintained at 24C throughout the test

pH:

pH at 0 hours in control and test material solutions ranged from 7.1 to 7.8. pH values at 72 hours in control and test material solutions ranged from 7.3 to 8.0.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks were used. Three flasks each containing 100 mL of test preparations were used for the control and each treatment group. The flasks were plugged with polyurethane foam bungs and incubated at 24 C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.

- Initial cells density: Preculture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.78 x10^6 cells per mL. Inoculation of test medium gave an initial cell density of 10^4 cells per mL.

- Control end cells density: Mean cell density of control at 0 hours was 1.60X10^4 cells per mL
Mean cell density of control at 72 hours was 1.37 x10^6 cells per mL

- No. of vessels per concentration (replicates): triplicate
- No. of vessels per control (replicates): triplicate

GROWTH MEDIUM
The culture medium used for the range finding and definitive tests was the same as that used to maintain the stock culture. Stock solutions of the culture medium was prepared using reverse osmosis purified deionized water. The culture medium contained the following macronutrients: NaNO3, MgCl2.6H2O, CaCl2.2H2O, MgSO4.7H2O, K2HPO4, NaHCO3. The culture medium contained the following micronutrients: H3BO3, MnCl2.4H2O, ZnCl2, FeCl3.6H2O, CoCl2.6H2O, Na2MoO4.2H2O, CuCl2*2H2O; Na2EDTA.2H2O, Na2SeO3.5H2O

- Standard medium used: yes

- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Light intensity and quality: 7000 lux continuous illumination

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Samples were taken at 1, 24, 48, and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.

TEST CONCENTRATIONS

- Range finding study: The loading rates to be used in the definitive test were determined by a preliminary range finding test. The range finding test was conducted by exposing Scenedesmus subspicatus cells to a series of nominal loading rates of 10, 100, and 1000 mg/L for a period of 72 hours. The test was conducted in 250 mL glass conical flasks plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. The test material was prepared as a WAF. After addition of the test material, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. These were stirred for 24 hours. The stirring was stopped and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that dispersed test material was present in the water column and hence it was considered justifiable to remove the undissolved material by filtering through a glass wool plug (2-4 cm in length). Microscopic observations of the WAFs performed after filtering showed that there were no globules/microdispersions of test material to be present.

The cell densities and percentage inhibition of growth values from the exposure of the alga to the test material during the range finding test showed no effect on growth at 10 mg/L loading rate WAF, however growth was observed to be reduced at 100 and 1000 mg/L loading rate WAFs. Based on this information and in order to ensure that both EL50 and NOEL values were obtained, loading rates of 10, 32, 100, 320, and 1000 mg/L using a stirring period of 24 hours followed by 1 hour standing period were selected for the definitive test. Due to a technical error the 100 mg/L loading rate WAF was incorrectly prepared resulting in a 220 mg/L loading rate WAF being used.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

410 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 320 - 520 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

240 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes

- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 10, 32, and 220 mg/L loading rate WAFs, however few intact cells were observed to be present in the test cultures at 320 and 1000 mg/L loading rate WAF.

Any other information on results incl. tables

Table. Inhibition of Growth Rate and Biomass

Nominal Loading Rate (mg/L)	Area Under Curve at 72 h	% Inhibition	Growth Rate (0-72 h)	% Inhibition
Control	2.82E7	--	0.062	--
10	2.61E7	7	0.065	[5, increase compared to controls]
32	2.46E7	13	0.057	8
220	2.65E7	6	-0.064	[3, increase compared to controls]
320	7.72E5	97	-0.016	74
1000	-4.09E5	101	-0.012	119

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Scenedesmus subspicatus to the test material gave an EbL50 (72h) value of 240 mg/L loading rate WAF and an ErL50 (0-72hr) value of 410 mg/L loading rate WAF. The No Observed Effect Loading rate was 220 mg/L loading rate WAF.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green algaScenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No. 201, “Alga Growth Inhibition Test”. 

 

Methods:

Following a preliminary range finding test, the alga were exposed to Water Accommodated Fractions of test material over a range of nominal loading rates of 10, 32, 220, 320, and 1,000 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 degrees C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using a Coulter Multisizer Particle Counter.

 

Results:

Exposure ofScenedesmus subspicatusto the test material gave an EbL50 (72h) value of 240 mg/L loading rate WAF and an ErL50 (0-72hr) value of 410 mg/L loading rate WAF. The No Observed Effect Loading rate was 220 mg/L loading rate WAF.