O,O,O-triphenyl phosphorothioate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Effect concentrations are not reported as time-weighted mean although measured test concentrations were not within the range of 80-120%. The study was conducted with an UVCB which O,O,O-triphenyl phosphorothioate is one of the main constituents of.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration of the test substance was determined in one representative test interval per week in each freshly prepared test solution (test vessels without daphnids) and in the 24h old test solutions before renewal (combined sample from test vessels with daphnids).
Additionally, retained samples were collected from the 24 hour old abiotic replicate (no Daphnia or food algae) per test group.
Samples were taken on study day 1, 2, 8, 9, 17, 18.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is a UVCB substance which is sparingly soluble in water . Therefore the test solution was prepared following general guidance provided in OECD 23 in order to generate a water accommodated fraction of the test substance in test medium.
- Method: The test solution was prepared by directly adding test substance to test medium according to
the loading rate (11mg test substance in 2000mL test medium for limit concentration of 5.5mg/L). The mixture was stirred on a magnetic stir plate for about 3 days at room temperature. After stirring, the mixture was left undisturbed for about 1 hour to allow separation of the undissolved material. The test substance density is >1 and droplet balls were observed on the bottom of the flask. The aqueous fraction was drawn off and any remaining undissolved test substance was removed by filtration (Schott glass filtration unit with 0.2 μm nylon membrane, Whatman) prior to testing. Filters were wetted and rinsed with at least 100 mL of highly distilled water and some residual water was left standing over the filter to prevent drying out. Approximately 500 mL of the test solution was added to the filtration unit and the first 50–90 mL were discarded in order to saturate the filter. The remainder of the test solution was bulk filtered to the point where approximately 10 mL remained standing over the filter. The filtered test solution was used for toxicity testing. Control test media was similarly filtered. Fresh test solutions were prepared daily up to day 20.
Although there was no evidence to suggest exposure concentration instability, fresh test solutions were prepared daily as a precautionary measure to help maintain consistent exposure to the test substance.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
The exposure was started after separation of undissolved material. The loading refers to all soluble components of the original nominal concentration. All test solutions were visibly colorless and clear throughout each renewal period.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna STRAUS 1820
- Source: supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Culture consitions: Daphnia brood stock are kept in mass cultures consisting of approx. 20–30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at test initiation. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are used for testing:
- Age at test initiation: < 24h


ACCLIMATION
-The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ±1°C), and diet.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

22 d

Hardness:

2.38 - 2.47 mmol/L

Test temperature:

20-21°C
with the exception of study day 20-22 where the temperature increased from 21.6-21.9°C. This increase of the temperature was due to a malfunction of the technical system. However this small increase is not expected to influence the result of the study.

pH:

7.8 - 8.2

Dissolved oxygen:

4.7 - 8.9
The low dissolved oxygen concentrations were due to a degassing of the test solution during filtration.

Nominal and measured concentrations:

5.5mg/L loading rate ( WAF),
Peaks corresponding to the test substance were measured in many of the samples from the treatment group, but several were The measured concentrations do confirm exposure to the dissolved test substance components in the WAF, but should not be used as a basis to set effect concentrations due to the high variability.

Details on test conditions:

TEST SYSTEM
- Test vessel: Numbered glass beakers (nominal volume 100 mL), covered with glass Petri plates to slow evaporation, filled with 50ml test volume
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): Semi-static, daily renewal (except study days 21-22 when the daphnids remained in the static test solution for 48hours).
- No. of organisms per vessel: 1 animal (0.02 animals/ mL)
- No. of vessels per concentration (replicates): 10 replicates (1 parent animal each)
- No. of vessels per control (replicates): 10 replicates (1 parent animal each)
- Food: During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium. The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 1300 mg and 1760 mg TOC/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤154 μL) of the concentrate to reach the
amount of food defined in the table below. The algae were stored in a refrigerator (dark, about 4-8°C) for maximum 21 days. By adding the algal concentrate the test solution was slightly diluted. During a 24 h test interval with 1 feeding, a total of 0.154 mL are added to 50 mL test volume
resulting in a maximal dilution of 0.3%.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 211. (Total hardness: 2.20 – 3.20 mmol/L, Acid capacity up to pH 4.3: 0.80 – 1.00 mmol/L, pH value: 7.5 – 8.5, Dissolved oxygen: Must remain ≥ 3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 h prior to use.)
- Total organic carbon: <2 mg/L
- Ca/mg ratio: about 4 : 1
- Conductivity: 550-650 μS/cm
- Intervals of water quality measurement:
--daily: appearance of the test solutions and dissolution behavior of the test substance was observed and recorded
-- after aeration and prior to use: chemical and physical parameters of the test medium (total hardness, acid capacity, pH, conductivity and total organic carbon)
--Dissolved oxygen, pH and temperature were measured in the old and in the freshly prepared test solution in replicate 1 of each test group for one interval per week. In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels. Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod/ - Light intensity: 649–691 lux at a wave length of 400–750 nm. The light intensity will not exceed 1000–1500 lux for cool white light (equivalent to 15-20 μE•m-2•s-1 according to the test guideline). 16 hours light : 8 hours darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Every day: Parent mortality, abnormal effects, and numbers of live and dead offspring
- Reproductive success: counting, discarding the offspring produced by each parent for the duration of the study. (Separation of neonates by wider pore pipet)
- prolonged exposure: On day 21 of exposure, some replicates had not produced a complete final brood. Therefore the test was prolonged an extra day to allow Daphnia in all replicates to release their final brood. At test termination, the surviving adults were removed from the test vessels and
euthanized by immersion in 70% isopropanol. Individuals were then isolated on a labeled glass slide. The body length of each adult daphnid was measured from the apex of the helmet to the base of the posterior spine (i.e. excluding the anal spine and protruding second antennae) with a binocular dissecting microscope (Leitz Diavert) and a calibrated eyepiece micrometer which was checked with a standardized slide.


RANGE-FINDING STUDY
In an acute Daphnia study no immobilization was observed at a loading rate of 100 mg/L (information from the sponsor). Therefore the chronic Daphnia study was performed as a limit test. The test substance is poorly water soluble and a UVCB substance. According to the OECD 211 guideline, the highest suggested test concentration for a limit test is 10 mg/L or the saturation limit in test medium, whichever is lower. The loading rate in this study was selected in agreement with the sponsor to achieve a saturated water accommodated fraction (WAF) of the test substance.

Reference substance (positive control):

yes

Remarks:

sodium chloride

Duration:

22 d

Dose descriptor:

NOELR

Effect conc.:

> 5.5 mg/L

Nominal / measured:

nominal

Basis for effect:

reproduction

Duration:

22 d

Dose descriptor:

LOELR

Effect conc.:

>= 5.5 mg/L

Nominal / measured:

nominal

Basis for effect:

reproduction

Details on results:

There was no significant adverse effect on survival or reproduction in the treatment group after 22 days. The data were not sufficient to calculate ECx values for reproduction.
No additional adverse effects or abnormal behavior were observed in any of the test groups.

All test solutions were visibly colorless and clear throughout each renewal period. No undissolved test substance or other unusual manifestations were observed.

The test substance had no observable chronic effect on Daphnia magna up to its saturation limit in test media and under test conditions.

The test substance is a sparingly water soluble UVCB and the treatment group was prepared using a loading rate far in excess of the reported water solubility (approx. 0.1 mg/L for the main component) to generate a water accommodated fraction (WAF) of the test substance. The analytical concentration control was based on the measurement of the two strongest peaks. Results were inconsistent and varied widely. The measured concentrations do confirm exposure to the dissolved test substance components in the WAF, but should not be used as a basis to set effect concentrations due to the high variability. Nevertheless all reasonable efforts were taken to produce a saturated WAF of all soluble components of the test substance in test media. The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. According to OECD 23 [Ref. 8], for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or loading rate (for mixtures).

Results with reference substance (positive control):

acute toxicity tests with a reference substance are conducted monthly. Reference substance tests are conducted generally according to the OECD 202 guideline and in accordance with GLP, but without a GLP status. The EC50(48 h) of the reference substance sodium chloride (NaCl) was 4.99 g/L (experiment date: 05 Aug 2014, project number: 50E0789/12E046). This result is within the range of 3.88–7.22 g/L, which represents ±2 standard deviations from the published EC50(48 h) of 5.55 g/L [Ref. 6, 7] and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

No results available yet as the study is still ongoing

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 May 2021 to 26 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, Treatment

- Sampling method: The concentration of the test substance was determined in each freshly prepared test solution (test solution tank without daphnids) and in the 48 hour or 72 hour aged test solutions before renewal (combined biotic) and in the 48 hour or 72 hour aged test solution before renewal (combined abiotic). When samples were combined, they were filled in 24h preconditioned vessels of sufficient volume. For biotic samples, all 10 replicates were combined and for abiotic samples all three replicates were combined. The samples (exactly 50 mL/sample) were taken from the combined test solution from all test group replicates using a glass pipette and were stored at the Laboratory ECO in a freezer (at – 20 °C) or transported to the analytical laboratory. Samples were transported to the analytical laboratory fresh or frozen (depending on capacities in analytical laboratory) in glass flasks or similar. Samples were taken on Day 0, 2, 5, 7, 9, 12, 14, 16, 19 and 21.

- Sample storage conditions before analysis: Depending on the capacities of the analytical laboratory, the samples were freshly analyzed or stored at -20 °C before analyzing.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (100% saturated) was prepared using a solid-liquid (slow-stir) saturator technique.This technique involves coating the bottom surface of a stainless steel stock solution tank with the test substance to generate a saturated aqueous solution. To facilitate substance handling, the test substance was weighed (2 mg) and then dissolved in 100 mL acetone. The acetone solution was checked for complete dissolution of the test substance, then 10 mL of the acetonic solution was distributed on the bottom of the stock solution tank. All the acetone was then completely evaporated to leave a thin layer of test substance coating the bottom of the stock solution tank. Afterwards 10 L of M4 dilution water was added (paying attention to minimize the disturbance of the test substance layer) and the stock solution (approx. 0.02 mg/L target concentration) was stirred with a paddle stirrer at the test temperature for approx. 24 hours. Since all acetone was evaporated prior to adding water, no solvent was present in the final test solution and no solvent control was required. Control was treated in the same way without application of test substance

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions were visibly colorless and clear throughout each renewal period. Only once undissolved test substance was observed on the surface, but test solution was taken from the middle of the tank (no particles observed and Tyndall negative), and since measured concentration was in the range of previous measured concentrations this single observation did not influence the outcome of this study.

- Other relevant information: To limit the potential for test substance loss by binding to test vessels, all test vessels were soaked for approximately 1 day with the saturated test solution prepared as described above. The old solution was discarded, and fresh solution was filled for testing in the pre-soaked test vessels. For each test solution renewal a new stock solution was used and the test vessels used were pre-conditioned for approx. 1 day with the same stock solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna STRAUS
- Age at study initiation: < 24h
- Weight at study initiation:
- Length at study initiation (length definition, mean, range and SD):
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978.
From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Feeding during test
During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium. The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 1660 mg and 1760 mg TOC*/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤120 µL) of the concentrate to reach the amount of food defined in the table below. The algae were stored in a refrigerator (dark, about 4 8°C) for maximum 21 days.
By adding the algal concentrate, the test solution was slightly diluted. During a 72 h test interval with 3 feedings, 0.12 mL each, a total of 0.36 mL are added to 50 mL test volume resulting in a maximal dilution of 0.72%.

Day of Test Amount of food per parent animal and day [mg TOC] *
0-3 0.10
4-5 0.12
6-7 0.14
8-test end 0.20


The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ±1°C), and diet.

Daphnia brood stock are kept in mass cultures consisting of approx. 20–30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at start of exposure. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are used for testing.

Test type:

semi-static

Water media type:

freshwater

Remarks:

A synthetic fresh water (Elendt M4) is used as media for culture and test purposes.

Limit test:

yes

Total exposure duration:

21 d

Hardness:

2.20 - 2.21 mmol/L

Test temperature:

19.5 - 20 °C

pH:

8 - 8.2

Dissolved oxygen:

8.7 - 10.4 mg/L

Conductivity:

550 - 650 µS/cm

Nominal and measured concentrations:

Mean initial measured saturation concentration: 7.04 µg/L
Time-weighted mean: 7.24 µg/L

Measured values were lower in old biotic test solutions and exceeded >20% of nominal. Since
measured values in abiotic samples deviated less than 20% from the initial measured
saturation concentration of each renewal period (with exception of one sample at 79% of the
initial measured saturation concentration), those were considered to represent the actual
exposure concentration. These low measured values are most likely due to the test substance
binding to the increased amount of food (algal cells) used in the later days of the test.
Therefore, only measurements of fresh and aged abiotic samples were used for calculation of
the effect concentrations. All measured concentrations in the abiotic samples deviated less than 20% from the initial
measured saturation concentration of each renewal period with exception of one sample on
study day 5 which was at 79% of the initial measured saturation concentration.
However, two of the initial measured saturation concentrations varied slightly more than >20%
from the mean measured initial saturation concentration and therefore the time-weighted mean
was considered to be an accurate representation of exposure levels maintained throughout
the test period. Following recommendations in OECD 211 the results should be evaluated
based on the time weighted mean measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (100 mL) covered with glass petri plates to slow evaporation
- Material, size, headspace, fill volume: Test volume 50 mL
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): 48 - 72 h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 0.02 animals/mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic fresh water Elendt M4
- Total organic carbon: < 2 mg/L
- Ca/mg ratio: about 4:1
- Conductivity: 550-650 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, conductivity and total organic carbon) were determined after aeration and prior to use in the test and were within acceptable ranges. Dissolved oxygen, pH and temperature were measured in the old and in the freshly prepared test solution in one replicate of each test group for one interval per week. In addition temperature was measured continously during the whole exposure period in a seperate vessel. Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 light/dark
- Light intensity: 683-781 lux at 400 - 750 nm

EFFECT PARAMETERS MEASURED:
- Parent mortality, abnormal effects, and numbers of live and dead offspring were assessed every day throughout the experiment. Reproductive success was measured by counting and
discarding the offspring produced by each parent / replicate for the duration of the study.
- Other biological observations were Mean Growth, % Immobile Young, Mean Days to First Brood and % Aborted Eggs

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: Substance is poorly water soluble and was tested at saturation concentration
- Range finding study: Preliminary test at saturation concentration
- Results used to determine the conditions for the definitive study: No effects observed in preliminary test

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 7.24 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 7.24 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 7.24 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 7.24 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

No mortality was observed among parent animals over the 21d exposure period.
There was no signifcant adverse effect on Daphnia reproduction or survival in the treatment group at the calculated time-weighted mean concentration of 7.24 µg/L.
No additional adverse effects or abnormal behavior were observed in any of the test groups.

Results with reference substance (positive control):

In order to verify that the Daphnia magna culture is responding normally to toxic stress, acute toxicity tests with a reference substance are conducted monthly. Reference substance tests are conducted generally according to the OECD 202 guideline and in accordance with GLP, but without a GLP status.
The EC50(48 h) of the reference substance sodium chloride (NaCl) was 5.17 g/L (experiment date: 21 Apr 2021, project number: 50E0789/12E067). This result is within the range of 3.88–7.22 g/L, which represents ±2 standard deviations from the published EC50(48 h) of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

Reported statistics and error estimates:

- Reproduction as number of living young (Wilcoxon’s test, one sided analysis)
- % immobile young (Wilcoxon’s test, one sided analysis)
- % aborted eggs (Wilcoxon’s test, one sided analysis)
- Time to first brood (Wilcoxon’s test, one sided analysis)
- Growth as length in mm (Student´s t-test, one sided analysis).

Effect concentrations (ECx) could not be calculated since there were no effects at the tested
concentrations.

Test group [% saturation concentration]	Mean Growth [length, mm]	% Immobile Young	Mean Days to First Brood	% Aborted Eggs
0	4.27	0	8.9	0
100	4.28	0	9.5	0

 

Test Group [% saturation concentration]	Reproduction		Mortality
	Mean Living Young per surviving adult	% effect	Parent animals	% effect
0	124	-	0	-
100	126	-	0	-

Validity criteria fulfilled:

yes

Conclusions:

No adverse chronic effects of O,O,O-Triphenyl phosphorothioate on Daphnia magna were observed up to the limit of water solubility.

Executive summary:

In a 21-day chronic toxicity study according to OECD 211, Daphnia magna were exposed to O,O,O-Triphenylphosphorothioate at a saturation concentration of 0 (control) and 100% under static renewal conditions in accordance with the OECD 211 guideline. The water pH, temperature and dissolved oxygen were within acceptable guideline specifications. Mortality, reproduction and sublethal effects were observed daily. No significant mortality or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. The data were not sufficient to calculate ECx values for reproduction or mortality.
The test substance did not have a chronic toxic effect on Daphnia magna up to the saturation concentration of 7.24 μg/L.

Description of key information

The substance is not harmful to aquatic invertebrates at chronic exposure up to the limit of solubility in water.

Additional information

In a key study investigating the long-term toxicity to aquatic invertebrates according to OECD 211 (BASF, 2021) O,O,O-triphenyl phosphorothioate (CAS 597-82-0) was tested at a saturation concentration of 7.24 µg/L. No significant effect on parent mortality or reproduction or any other additional significant effects or abnormal behavior were observed in any of the test treatments. The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

In a supporting chronic toxicity test on Daphnia magna reproduction according to OECD 211, O,O,O-triphenylphosphorotioate in a lower purity (purity > 25%) was investigated at saturation concentration(BASF 2015). As the test substance is a UVCB substance which is sparingly soluble in water, the test solution was prepared following general guidance provided in OECD 23 in order to generate a water accommodated fraction of the test substance in test medium (undiluted filtrate up to a loading rate of 5.5 mg/L). This substance is a complex substance containing as major component CAS 597-82-0 > 25%. As no adverse effects were observed up to the highest tested applied concentration it can be assumed that also CAS 597-82-0 does not cause adverse effects at saturation concentration below or equal to a loading rate of 1mg/L, correspondingly.

An older study on chronic toxicity to Daphnia (Wildlife International, 2003) with O,O,O-triphenylphosphorotioate in a lower purity (purity > 25%) is available and was re-evaluated for validity and considering the latest recommendations of OECD No. 23: Guidance Document on the Aquatic Toxicity Testing of Difficult Substances and Mixtures it is concluded that the study suffers from major flaws which are related to improper preparation of the test solutions resulting in the presence of undissolved test substance in the test vessels. Since the test item (EC 421-820-9) is a multi-constituent substance, test solutions for aquatic ecotoxicity tests should be prepared as water accommodated fractions (WAF) as recommended by OECD No. 23: Guidance Document on the Aquatic Toxicity Testing of Difficult Substances and Mixtures (the available acute tests on fish, Daphnia and algae were prepared as WAF solutions resulting in NOECs > 100 mg/L). In the present test, the test solutions were prepared as dilutions of a solvent stock solution. Measured concentrations of the stock solutions were 94 -97% of nominal but the measured test solutions were 38 -88% of nominal. This level of variability in the analytical results is typically indicative of undissolved substance. The report also confirms the visual presence of undissolved substance in the diluters. The OECD No. 23 guidance document explicitly discourages both the use of solvents and serial dilution when testing multi-component substances. Therefore, the study is not reliable and was not further considered in the ecotoxicological risk assessment. Further both supporting studies were conducted with a multi-constituent substance which can merely serve as an indication of chronic toxicity of O,O,O-Triphenyl phosphorothioate.