2-tetradecyloxirane, reaction products with boric acid

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:

21-10-2002 to 29-01-2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

no data about batch no. and purity of the test material

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

not applicable

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test material (10, 32, 100, 80 and 250 mg) were each separately added to the surface of 10, 10, 10, 2.5 and 2.5 litres of reconstituted water respectively to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates. After the addition of the test material, the reconstituted water was stirred for 24 hours by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. The stirring was stopped after 24 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Parafilm was submerged into each vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Parafilm seal. The aqueous phases or WAFs were removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Visual inspection of the WAFs showed undissolved test material to be present in them; therefore a glass wool plug was used to filter the WAFs prior to use.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea (Daphnia)
- Strain: Daphnia magna
- Source: In-house laboratory cultures
- Age of parental stock: 1st instar (<24 hours old)
- Feeding during test: Yes
- Food type: Unicellular algal culture (Chlorella sp.)
- Amount: 1.5-8.0 µL/daphnid; equivalent to 0.1-0.2 mg carbon/daphnid/day
- Frequency: Daily

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 21°C. 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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

220-265 mg/L as CaCO3

Test temperature:

18.8-20.8 °C

pH:

7.7-8.1

Dissolved oxygen:

8.2-8.7 mg O2/L

Salinity:

Not applicable. Study was performed in fresh water.

Nominal and measured concentrations:

Nominal loading rates: 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass flasks containing 100 mL of the test preparation
- Type: Covered- Aeration: The test vessels were not aerated. The diluent water only was aerated (prior to use) until the dissolved oxygen concentration was approximately air-saturation value.
- Renewal rate of test solution (frequency/flow rate): 3 times per week on Days 2, 5, 7, 9, 12, 14, 16 and 19
- No. of organisms per vessel: Single
- No. of vessels per concentration (replicates): 10- No. of vessels per blank control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water
- Conductivity: < 5 µS/cm

EQUIPMENT:
- Young daphnids (live and dead) and any unhatched eggs were counted using a stereo microscope.
- The pH was measured using a WTW pH 330 pH meter, the dissolved oxygen concentration was measured using a YSI 54A dissolved oxygen meter and the temperature was measured using a Multi-Stem ST-9269A digital thermometer.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the reconstituted water was adjusted (if necessary) with NaOH or HCl.
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 329-422 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- On a daily basis the numbers of live and dead of the Parental (P1) generation, the numbers of live and dead Filial (F1) Daphnia and the number of discarded unhatched eggs were counted.
- An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
- The number of Daphnia with eggs or young in the brood pouch was determined daily.
- Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination.
- Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (i.e. immobile) were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
- At the end of the test, the length of each surviving parent animal was determined.

BLANK CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L (nominal loading rates)
- Results used to determine the conditions for the definitive study: In the preliminary range-finding test, no immobilisation was observed at the test loading rates of 1.0 and 10 mg/L. However, immobilisation was observed at the 100 mg/L loading rate WAF. Based on these results, doses were assigned to the definitive test.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

20 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95% CL = 13-31 mg/L

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

23 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL = 17-30 mg/L

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

32 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

- Mortality of parent animals: 0, 0, 0, 10, 80 and 100% at 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L, respectively; effect was highly significant at 32 and 100 mg/L - Mean no. of offspring produced per female (cumulative): 78, 76, 76, 77 and 14 at 0 (control), 1.0, 3.2, 10 and 32 mg/L, respectively; effect was significant at 32 mg/L- Body length of parent animals: No statistically significant differences in length of daphnids were found between the control and treatment groups.- Type and number of morphological abnormalities: Daphnids in 32 and 100 mg/L group were significantly smaller and/or paler in colour than the control animals.- Egg development time: There were no unhatched eggs and dead young in all control and treatment groups surviving to maturation.- The 7, 14 and 21-Day EL50 (immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (PI) were calculated to be 44 (26-83), 31 (19-50) and 20 (13-31) mg/L loading rate WAF, respectively.- The 21-Day EL50 (reproduction) value based on nominal test concentrations was calculated to be 23 (17-30) mg/L loading rate WAF. - The Lowest Observed Effect Loading rate (LOEL) was considered to be 32 mg/L loading rate WAF on the basis that at this loading rate significantly fewer live young per adult (P<0.05) were produced when compared to the control and significant mortalities were observed in the parental generation (PI). Given that there was an 83% reduction in numbers of young produced per live adult compared to the control group and 80% mortality was observed at the LOEL, the nature of these data gives Day 14 and Day 21 EL50 (immobilisation) values and a Day 21 EL50 (reproduction) value of less than the LOEL.- The No Observed Effect Loading rate (NOEL) was considered to be 10 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 10 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

- The EL50 (immobilisation) values and associated confidence limits at 7, 14 and 21 days were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).- The observed mortality in the parental (P1) generation of the 10 mg/L test group was compared to the control group using the corrected chi-squared statistic (Breslow and Day 1980).- The EL50 (reproduction) value and associated confidence limits after 21 days were calculated by the trimmed Spearman-Karber method (Hamilton et al., 1977) using the ToxCalc computer software package (ToxCalc 1999).- For the estimation of the LOEC and NOEC, daphnia length data and the numbers of live young produced per adult over the duration of the test for the control 1.0, 3.2, 10 and 32 mg/L loading rate WAF test groups were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955).- All statistical analyses were performed using the SAS computer software package (SAS 1999-2001).

None

Validity criteria fulfilled:

yes

Remarks:

no mortality in control group; dissolved oxygen ≥ 8.1 mg O2/l; pH 0.2; mean no. of live young per surviving adult (control group) was 78 after 21 days; coefficient of variation for control group was 8%

Conclusions:

Under the test conditions, exposure of Daphnia magna to the test material resulted in significant mortalities at the loading rates of 32 and 100 mg/L resulting in 80% and 100% mortalities by Day 21 respectively. The 7, 14 and 21-Day EL50 (immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (PI) were calculated to be 44 (26-83), 31 (19-50) and 20 (13-31) mg/L loading rate WAF, respectively. A significant impairment of reproduction was observed at the 32 mg/L loading rate WAF test group after 21 days. The 21-Day EL50 (reproduction) value based on nominal test concentrations was calculated to be 23 (17-30) mg/L loading rate WAF.The LOEL and the NOEL based on nominal loading rates were 32 and 10 mg/L respectively.

Executive summary:

Introduction

A study was performed to assess the effect of the test material on the reproduction of Daphnia magna over a 21-day period. The method followed that described in the OECD Guidelines for Testing of Chemicals No 211 (1998) "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Directive 2001/59/EC (which constitutes Annex V of Council Directive 67/548/EEC).

 

Methods

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test material over a range of loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days. The WAFs were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension.

 

Results

The 7, 14 and 21-Day EL50 (immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (PI) were calculated to be 44 (26-83), 31 (19-50) and 20 (13-31) mg/L loading rate WAF, respectively.

The 21-Day EL50 (reproduction) value based on nominal test concentrations was calculated to be 23 (17-30) mg/L loading rate WAF.

The Lowest Observed Effect Loading rate (LOEL) was considered to be 32 mg/L loading rate WAF on the basis that at this loading rate significantly fewer live young per adult (P<0.05) were produced when compared to the control and significant mortalities were observed in the parental generation (PI). Given that there was an 83% reduction in numbers of young produced per live adult compared to the control group and 80% mortality was observed at the LOEL, the nature of these data gives Day 14 and Day 21 EL50 (immobilisation) values and a Day 21 EL50 (reproduction) value of less than the LOEL.

The No Observed Effect Loading rate (NOEL) was considered to be 10 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 10 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

 

Conclusion

Under the test conditions, exposure of Daphnia magna to the test material resulted in significant mortalities at the loading rates of 32 and 100 mg/L resulting in 80% and 100% mortalities by Day 21 respectively. The 7, 14 and 21-Day EL50 (immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (PI) were calculated to be 44 (26-83), 31 (19-50) and 20 (13-31) mg/L loading rate WAF, respectively. A significant impairment of reproduction was observed at the 32 mg/L loading rate WAF test group after 21 days. The 21-Day EL50 (reproduction) value based on nominal test concentrations was calculated to be 23 (17-30) mg/L loading rate WAF.

The LOEL and the NOEL based on nominal loading rates were 32 and 10 mg/L respectively.

Description of key information

The 21-Day EL50 (reproduction) value based on nominal test concentrations was calculated to be 23 (17-30) mg/L loading rate WAF. The LOEL and the NOEL based on nominal loading rates were 32 and 10 mg/L respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10 mg/L

Additional information

A key study to assess the effect of the test material on the reproduction of Daphnia magna over a 21-day period was performed to OECD Guideline No 211.

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test material over a range of loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days. The WAFs were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension.

Under the test conditions, exposure of Daphnia magna to the test material resulted in significant mortalities at the loading rates of 32 and 100 mg/L resulting in 80% and 100% mortalities by Day 21 respectively. The 7, 14 and 21-Day EL50 (immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (P1) were calculated to be 44 (26-83), 31 (19-50) and 20 (13-31) mg/L loading rate WAF, respectively. A significant impairment of reproduction was observed at the 32 mg/L loading rate WAF test group after 21 days. The 21-Day EL50 (reproduction) value based on nominal test concentrations was calculated to be 23 (17-30) mg/L loading rate WAF. The LOEL and the NOEL based on nominal loading rates were 32 and 10 mg/L respectively.