Reaction products of bis(4-methylpentan-2-yl)dithiophosphoric acid with phosphorus oxide, propylene oxide and amines, C12-14-alkyl (branched)

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 22 April 2008 and 11 June 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study; well-documented study report. Restriction due to lack of test material analyses but are not expected to have a major impact on the test result.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Analytical monitoring:

no

Details on sampling:

No data available.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test material was prepared as a water accommodated fraction (WAF).
- Eluate: No.
- Differential loading: amounts of test material (2.4, 7.4, 24, 74 and 120 mg) were each separately added to the surface of 20, 20, 20, 20 and 10 liter of reconstituted water respectively to give the 0.12, 0.37, 1.2, 3.7 and 12 mg/L loading rates. Fro the 2.4 mg (and 7.4 mg on a single occation), the test material was smeared onto a glass slide which was then suspended in the medium due to the small amount being weighed out. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such as a vortex was formed to give a slight dimple at the water surface. These were stirred fro 23 h. The stirring was stopped after 23 h and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into each vessel, sealed end down, to a depth of approximately 5cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phases or WAFs were removed by mid-depth siphoning to give the 0.12, 0.37, 1.2, 3.7 and 12 mg/L loading rates WAFs.
- Controls: negative control.
- 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): microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea
- Strain/clone: None stated.
- Justification for species other than prescribed by test guideline: NA.
- Source: Daphnia magna derived from in-house laboratory cultures.
- Age of parental stock (mean and range, SD): Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
- Feeding during test
- Food type: suspension of algae (Chlorella sp.).
- Amount: 5 to 10 ul unicellular algal culture per daphnia per day. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependant on the age and size of the animals. Equal amounts of food were given to each daphnid.
- Frequency: daily

ACCLIMATION
- Acclimation period: None stated.
- Acclimation conditions (same as test or not): None stated.
- Type and amount of food: None stated.
- Feeding frequency: None stated.
- Health during acclimation (any mortality observed): None stated.

QUARANTINE (wild caught)
- Duration: NA
- Health/mortality: NA

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water at a temperature of approximately 20 oC. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

other: Other: renewed 3 times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19.

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not applicable.

Hardness:

112-160 mg/l as CaCO3

Test temperature:

21-22OC

pH:

7.9-8.8

Dissolved oxygen:

93%-100% (percentage fo Air Saturation Value.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentrations: 0.12, 0.37, 1.2, 3.7 and 12 mg/L loading rates WAFs

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): glass flasks which were then covered with a plastic lid to reduce evaporation.
- Material, size, headspace, fill volume: for each concentration a single daphnid was placed in 100 ml of the test preparation in 150 ml glass flasks
- Aeration: The test vessels were not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable.
- Renewal rate of test solution (frequency/flow rate): The test preparations were renewed 3 times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): not applicable.
- Biomass loading rate: no data available.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water. The reconstituted water had a pH of 7.8 f 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as
CaCO3.
- Total organic carbon: NDA.
- Particulate matter: NDA.
- Metals: NDA.
- Pesticides: NDA.
- Chlorine: NDA.
- Alkalinity: NDA.
- Ca/mg ratio: NDA.
- Conductivity: NDA.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: NDA.

OTHER TEST CONDITIONS
- Adjustment of pH: (if necessary) with NaOH or HCl
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition
- Light intensity: 536 to 563 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: no data available.
- Justification for using less concentrations than requested by guideline: loading rate fro the definitive test was based on data from ranging finding study.
- Range finding study
- Test concentrations: 1.0, 3.2, 10, 32, 100 mg/l
- Results used to determine the conditions for the definitive study: Based on the results of an acute toxicity test, Daphnia magna were exposed to an aqueous dispersion of the test material over a range of test concentrations of 0.12, 0.37, 1.2, 3.7, and 12 mg/l for a period of 21 days.

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

For P1 Generation

Remarks on result:

other: 0.51-1.6 mg/l

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

For F1 generation

Remarks on result:

other: 0.37-1.2 mg/l

Duration:

21 d

Dose descriptor:

other: NOEL

Effect conc.:

0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

for parental generation (P1)

Remarks on result:

other: This NOEL was for parental generation (P1)

Duration:

21 d

Dose descriptor:

other: NOEL

Effect conc.:

0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Based on numbers of live young produced per adult by Day 21

Details on results:

- Mortality of parent animals: 100% mortalities occurred at 3.7 and 12 mg/L groups, 70% mortality at 1.2 mg/L group, and 60% mortality at 0.37 mg/L group. No mortality was observed in each of the control and 0.12 mg/l test concentration.

- No. of offspring produced per day per female:
Neg. Control: 68 offspring / female
0.12 (mg/L): 67 offspring / female
0.37 (mg/L): 32 offspring / female
1.2 (mg/L): 34 offspring / female
3.7 (mg/L): 0 offspring / female
12 (mg/L): 0 offspring / female
After 21 days there were not statistically significant differences between the control and 0.12 mg/L loading rate WAF test group in terms of the number of live young produced per adult. The 0.37 and 1.2 mg/L loading rate WAF test groups showed a statistically significant difference from the control and the remaining test groups after 21 days in terms of producing fewer number of live young per adult. The 3.7 and 12 mg/L loading rate WAF test groups data were not included in statistical analysis as exposure to the test material eliminate all the daphnids prior to day 21 of the test.
- Body length and weight of parent animals:
Daphnia Lengths at day 21
Neg. Control: 5.0 ± 0.1 mm
0.12 (mg/L): 5.0 ± 0.1 mm
0.37 (mg/L): 4.5 ± 0.2 mm
1.2 (mg/L): 4.6± 0.2 mm
There was no significant effect on the size and color of the daphnids in that 100% of the surviving daphnids at 3.7 and 12 mg/L loading rate WAFs were markedly smaller and paler in color than the control groups. There was a significant effect on the color of the daphnids in that 50 and 71% of the surviving daphnia at 0.37 and 1.2 mg/L loading rate WAFs were markedly smaller and paler in color than the control animal by Day 21.
The daphnids at the remaining loading rates were observed to be the same size and color as the control animals.

- Type and number of morphological abnormalities: pale in color.
- Type and number of behavioural abnormalities: No.
- Number of males and females (parental): NO DATA AVAILABLE
- Time to first brood release or time to hatch: Day 7 of the test.
- Egg development time: NO DATA AVAILABLE
- Brood size: NO DATA AVAILABLE
- Time to sexual maturity: NO DATA AVAILABLE
- Type and magnitude of biochemical changes: NO DATA AVAILABLE
- Other biological observations: NO DATA AVAILABLE
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

No data available.

Reported statistics and error estimates:

Analysis of numbers of live young produced per adult:
Results from the control and 0.12, 0.37 and 1.2 mg/L test groups were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance and Dunnett’s multiple comparison procedure for comparing several treatments with a control.
The 3.7 and 12 mg/L test groups were not included in the statistical analysis as the effects of exposure eliminate all the daphnids prior to the termination of the test.
Analysis of mortalities in the P1 generation:
A corrected chi-square statistic was performed to show whether the mortality in the 0.37 and 1.2 mg/L groups observed during the test were statistically significant when compared to the control group.
Analysis of the daphnia length data:
Results from the control and 0.12, 0.37, and 1.2 mg/L test groups Daphnia length data determined for the surviving daphnids on termination of the test were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance and Dunnett’s multiple comparison procedure for comparing several treatments with a control.
The 3.7 and 12 mg/L test groups were not included in the statistical analysis as the effects of exposure eliminate all the daphnids prior to the termination of the test.

Observations on test material solubility:At the start of the mixing period all the WAFs were observed to have clear colorless water columns with oily globules of test material floating at the water surface. The 0.12 mg/L loading rate WAF was observed to be a clear, colorless water column with oily test material smeared onto a glass slide suspended in the water column. After 23 h stirring and a 1h standing period the WAF were observed to be clear and colorless solutions.Lethal Effects on the Parental Generation (P₁)

Mortality (immobilisation) occurred predominantly at the 3.7 and 12 mg/l loading rate WAF resulting in 100% maotality by days 15 and 7, respectively. 

Mortality was also observed at the 0.37 and 1.2mg/l loading rate WAF, but did not reach statistically significance compared to the control group.

No mortality occurred at the 0.12 mg/l loading rate WAF test group throughout the test.

There was significant effect on the size and color of the daphnia in that 100% of the surviving daphnia at 3.7 and 12 mg/l loading rate WAFs were markedly smaller and paler in color than the control groups.

There was significant effect on the color of the daphnia in that 50% and 71% of the surviving daphnia at 0.37 and 1.2 mg/l loading rate WAFs were markedly smaller and paler in color than the control groups by day 21.

The daphnids at the remaining loading rates were observed to be the same size and color as the control animals.

Sublethal Effects on the Parental Generation (P₁)

After 21 days there were no statistically significant differences between the control and 0.12 mg/l loading rate WAF test group in terms of number of live young produced per adult. The 0.37 and 1.2 mg/l loading rate WAF test groups showed a statistically significant difference from the control and the remaining test group after 21 days in term of producing fewer numbers of live young per adult.

After 21 days the length of surviving adults was not statistically significant between the control and 0.12 mg/l loading rate WAF test group.

Lethal Effects on the Filial Generation (F₁)

The filial daphnids produced by the 0.12, 0.37 and 1.2 mg/l loading rate WAF test groups were in the same general condition as the young produced by the controls over the duration of the test.

Young were first produced in the control test group on day 7 of the test.

Due to the toxic effect of the test material the parental generation of the 3.7 and 1.2 mg/l loading rate WAF test groups were eliminated prior to the production of young.

Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

The old test media preparations were observed to be green tinged due to the presence of algal cells used as feed for the dapnids.

 

Validity criteria fulfilled:

yes

Conclusions:

No Observed Effect Loading rate" (NOEL) was considered to be 0.12 mg/l loading rate WAF on the basis that at this loading rate there were no significant mortalities (immobilisation) observed in the parental generation (P1) and that there were no significant differences (P ≥ 0.05) between the control and the 0.12 mg/l loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

Executive summary:

A study was performed to assess the effect of the test substance on the reproduction of Daphnia magna over a 21-day period following the method described in OECD 211. The following results were obtained:

1. The 14 and 21-Day EL₅₀ (immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (P₁) were calculated to be 1.1 and 0.91 mg/l loading rate WAF with 95% confidence limits of 0.64-2.1 mg/l loading rate WAF and 0.51 - 1.6 mg/l loading rate WAF respectively.
2. The 21-Day EL₅₀ (reproduction) value based on nominal loading rates was calculated to be 0.66 mg/l loading rate WAF with 95% confidence limits of 0.37-1.2 mg/l loading rate WAF.
3. The "Lowest Observed Effect Loading rate" (LOEL) was considered to be 0.37 mg/l loading rate WAF on the basis that at this loading rate significantly (P 0.05) fewer young per adult were produced when compared to the control.
4. The "No Observed Effect Loading rate" (NOEL) was considered to be 0.12 mg/l loading rate WAF on the basis that at this loading rate there were no significant mortalities (immobilisation) observed in the parental generation (P₁) and that there were no significant differences (P ≥ 0.05) between the control and the 0.12 mg/l loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

Description of key information

The "No Observed Effect Loading rate" (NOEL) was considered to be 0.12 mg/l loading rate WAF on the basis that at this loading rate there were no significant mortalities (immobilisation) observed in the parental generation (P1) and that there were no significant differences (P ≥ 0.05) between the control and the 0.12 mg/l loading rate WAF test group in terms of numbers of live young produced per adult by Day 21 (OECD 211).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.12 mg/L

Additional information

A key study was performed to assess the effect of the test substance on the reproduction of Daphnia magna over a 21-day period following the method described in OECD 211. The 14 and 21-Day EL50 (immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (P1) were calculated to be 1.1 and 0.91 mg/l loading rate WAF with 95% confidence limits of 0.64-2.1 mg/l loading rate WAF and 0.51 - 1.6 mg/l loading rate WAF respectively. The 21-Day EL50 (reproduction) value based on nominal loading rates was calculated to be 0.66 mg/l loading rate WAF with 95% confidence limits of 0.37-1.2 mg/l loading rate WAF. The "Lowest Observed Effect Loading rate" (LOEL) was considered to be 0.37 mg/l loading rate WAF on the basis that at this loading rate significantly (P 0.05) fewer young per adult were produced when compared to the control. The "No Observed Effect Loading rate" (NOEL) was considered to be 0.12 mg/l loading rate WAF on the basis that at this loading rate there were no significant mortalities (immobilisation) observed in the parental generation (P1) and that there were no significant differences (P ≥ 0.05) between the control and the 0.12 mg/l loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.