Isooctadecanoic acid, reaction products with tetraethylenepentamine

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:

supporting study

Study period:

1 December 2002 to 23 December 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.

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)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Test solutions were prepared individually by adding the appropriate quantity of test material (10, 32, 100, 80 and 250 mg), to the surface of an appropriate amount of reconstituted water (10, 10, 10, 2.5 and 2.5L) to give the desired test concentrations (1.0, 3.2, 10, 32 and 100 mg/L loading rates).

After addition of the test material the test solutions were stirred for 24 hours using a magnetic stirrer such that a vortex formed which represented a slight dimple at the water surface. Following stirring for 24 hours, the mixtures were allowed to stand for 1 hour. A wide bore tube, covered at one end with paraffin was submerged into each vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of chemical resistant flexible tubing was inserted into the glass tube and pushed through the paraffin seal. The aqueous phases or WAFs were removed by mid-depth (the first 75 – 100 mL discarded) to give the desired loading rate WAFs. Microscopic inspection of the WAFs showed no microscopic dispersions or undissolved test material, therefore a glass wool plug was not used to filter the WAFs.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: laboratory cultures maintained at the testing facility.
- Feeding during test: yes.
- Food type: unicellular algal culture (Chlorella sp).
- Amount: 3.8 - 8.6 µL/daphnid (0.1 - 0.2 mg carbon/daphnid/day).
- Frequency: daily.

ACCLIMATION
- Acclimation conditions: same as test.
- Type and amount of food: suspension of algae (Chlorella sp).
- Feeding frequency: daily.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: 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 first instar daphnids 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

Post exposure observation period:

Test termination following 21 day exposure period.

Hardness:

248 - 262 mg/L (as CaCO3)

Test temperature:

20.1 - 20.8 ºC

pH:

7.8 - 8.2

Dissolved oxygen:

8.1 - 8.7 mg O2/L
82 - 98 % (air saturation value)

Nominal and measured concentrations:

0 (control), 1.0, 10, 100 mg/L loading rate WAF (nominal, range-finding test)
0 (control), 1.0, 3.2, 10, 32, 100 mg/L loading rate WAF (nominal, definitive test)

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass flasks.
- Type: closed (each flask was covered with a plastic lid to reduce evaporation).
- Fill volume: 100 mL
- Aeration: test solutions were not aerated during the study. The diluent water only was aerated prior to use.
- Renewal rate of test solution: test solutions were renewed 3 times per week on days 2, 4, 7, 9, 11, 14, 16 and 18. Renewal was performed by transferring live adult Daphnids to fresh media.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (see "Any other information on materials and methods incl. tables" for further information).
- Culture medium different from test medium: no.
- Hardness: The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
- Intervals of water quality measurement: temperature and light intensity were recorded daily throughout the test. Dissolved oxygen, pH and temperature were recorded before and after each test media renewal.

OTHER TEST CONDITIONS
- Adjustment of pH: the pH of the dilution was adjusted to pH 7.8 ± 0.2 with NaOH or HCl, if required.
- Photoperiod: 16 hours light / 8 hours dark with 20 minute dawn and dusk transition periods.
- Light intensity: 529 - 591 lux.

EFFECT PARAMETERS MEASURED: the numbers of live and dead of the parental generation, the numbers of live and dead "Filial" (F1) Daphnia and the number of discarded unhatched eggs were counted, daily. 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 with the exception of days 12 and 13. 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 (immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of offspring produced in the flask.
At the end of the test, the length of each surviving parent animal was determined.

VEHICLE CONTROL PERFORMED: yes.

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 1.0, 10, 100 mg/L loading rate WAF (nominal).
- Results used to determine the conditions for the definitive study: no immobilisation was observed at any of the loading rates. Based on these data, the following test concentrations were assigned for definitive testing: 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

EL50

Effect conc.:

72 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water accommodated fraction (WAF)

Basis for effect:

immobilisation

Remarks on result:

other: 52 - 100 mg/L (95% CL)

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water accommodated fraction (WAF)

Basis for effect:

immobilisation

Remarks on result:

other: 52 - 82 mg/L (95% CL)

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

57 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water accommodated fraction (WAF)

Basis for effect:

reproduction

Remarks on result:

other: 38 - 85 mg/L (95% CL)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water accommodated fraction (WAF)

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water accommodated fraction (WAF)

Basis for effect:

immobilisation

Details on results:

- Observations of Test Material Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the stirring periods the loading rates were observed to be clear, colourless water columns with oily slicks of test material at the water surface. After 24 hours stirring and a 1 hour standing period the loading rates were observed to remain clear, colourless water columns with oily slicks of test material at the water surface.
Microscopic inspection of the WAFs showed no micro-dispersions to be present. Therefore a glass wool plug was not used to siphon the WAF.
The old or expired test media were observed to have a green colouration due to the algal cells given as food for the daphnids.

- Lethal Effects on the Parental Generation
Mortality (immobilisation) occurred predominantly at the highest test loading rate of 100 mg/L loading rate WAF resulting in 80% mortality by day 21, indicating a prolonged toxic effect attributable to exposure of Daphnia magna to the test material.
Mortality was also observed at all of the test loading rates in 10%, 10% and 20% mortality at the 1.0, 3.2 and 10 mg/L loading rate WAF groups respectively by day 21. At the 32 mg/L loading rate WAF test group 40% mortality was observed during the test. However, statistical analysis of the mortality data using the corrected chi-squared statistic showed that the observed mortalities in each of these test groups were not significantly different (p ≥ 0.05) when compared to the control group which showed 20% mortality during the test. This is important to note when interpreting the NOEC value.

- Sub-Lethal Effects on the Parental Generation
There was a significant effect on size and colour of the daphnids in that 33% to 100% of the surviving daphnids on day 7 to day 21 at the test loading rate of 100 mg/L were markedly smaller and paler in colour than the control animals. The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals.
After 21 days there were no statistically significant differences between the control and the 1.0, 3.2, 10 and 32 mg/L loading rate WAF test groups in terms of the number of live young produced per adult.
After 21 days the length of each surviving adult was determined. The results showed that there were no significant differences (p ≥ 0.05) between the control and the 1.0, 3.2, 10 and 32 mg/L loading rate WAF test groups in terms of length of the daphnids after 21 days exposure to the test material.
The 100 mg/L loading rate WAF test group data was not included in the statistical analysis due to significant mortalities in the parental generation over the duration of the test.

- Effects on the Filial Generation (F1)
Information on the effects of the test material on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.
Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

Reported statistics and error estimates:

The EL50 (immobilisation) values and associated confidence limits at 14 and 21 days were calculated by the trimmed Spearman-Karber method (Hamilton at al 1977) using statistical software. This method is appropriate when only one partial response is shown.
The observed mortalities n the parental (P1) generation of each of the test groups 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 maximum-likelihood probit method (Finney 1971) again using statistical software. The probit analysis was used where two or more practical responses to exposure are shown.
The numbers of live young produced per surviving adult in the control were compared to those of the 1.0, 3.2 10 and 32 mg/L test groups using one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatment with a control. Comparisons were also made using a non-parametric analogue to one-way analysis of variance, the Kruskal-Wallis test.
Results on Daphnia length from the control and 1.0, 3.2, 10 and 32 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.
All statistical analyses were performed using a statistical computer software package.
The 100 mg/L loading rate test groups data were not included in the statistical analysis due to the observation of significant mortalities in the parental generation.

Table 1: Summary of Results

Nominal loading rate (mg/L)	% survival of P1	No. of live young*		No. of dead young*		No of unhatched eggs*
		Total	per female (cumulative)	Total	per female (cumulative)	Total	per female (cumulative)
0 (control)	80	914	100	0	0	1	< 1
1.0	90	904	98	0	0	0	0
3.2	90	885	98	0	0	0	0
10	80	757	95	0	0	1	< 1
32	60	661	92	0	0	0	0
100	20	34	11	0	0	0	0

* the numbers of live and dead young and unhatched eggs are cumulative totals.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, exposure of Daphnia magna to the test material resulted in significant mortalities at the test concentration of 100 mg/L loading rate WAF resulting in 80% mortalities by day 21. The 14 and 21 day EL50 (immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (P1) were determined to be 72 and 65 mg/L loading rate WAF, respectively. No significant impairment of reproduction was observed at the test concentrations employed during the test. The 21 day EL50 (reproduction), was determined to be 57 mg/L (nominal) loading rate WAF.
The Lowest Observed Concentration (LOEC) and the No Observed Effect Concentration (NOEC) were determined to be 100 mg/L and 32 mg/L loading rate WAF. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The toxicity of the test material to aquatic invertebrates was investigated in Daphnia magna in accordance with standardised guidelines OECD 211 and EU Method C.20. Based on the results of a preliminary range-finding test, the Daphnia were exposed to a Water Accommodated Fraction of 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 three time 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. The 14 and 21 day EL50 (immobilisation) values, based on nominal concentrations, for the parental Daphnia generation (P1) were determined to be 72 and 65 mg/L loading rate WAF, respectively. The 21 day EL50 (reproduction) was determined to be 57 mg/L loading rate WAF. The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) were determined to be 100 mg/L and 32 mg/L loading rate WAF, respectively. The NOEC was determined on the basis that at this test concentration there were no significant mortalities observed in the parental generation and there were no significant differences between the control and the 32 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by day 21. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Description of key information

21 d EL50 = 65 mg/L (immobilisation); 21 d EL50 = 57 mg/L (reproduction); 21 d NOEC = 32 mg/L, Daphnia magna, OECD 211, EU Method C.20, Sewell 2003

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

32 mg/L

Additional information

The toxicity of the test material to aquatic invertebrates was investigated in Daphnia magna in accordance with standardised guidelines OECD 211 and EU Method C.20. Based on the results of a preliminary range-finding test, the Daphnia were exposed to a Water Accommodated Fraction of 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 three time 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. The 14 and 21 day EL50 (immobilisation) values, based on nominal concentrations, for the parental Daphnia generation (P1) were determined to be 72 and 65 mg/L loading rate WAF, respectively. The 21 day EL50 (reproduction) was determined to be 57 mg/L loading rate WAF. The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) were determined to be 100 mg/L and 32 mg/L loading rate WAF, respectively. The NOEC was determined on the basis that at this test concentration there were no significant mortalities observed in the parental generation and there were no significant differences between the control and the 32 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by day 21. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes. The study was performed in line with GLP and accepted standardised guidelines and assigned a reliability score of 1.