p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

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:

20 September 2011 - 29 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10706: “Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea)”, 2000-03-30.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Volatile: No
Stability in water: No
Solubility in water: No

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the control. The method of analysis is described in the appended Analytical Report.
Sampling :
- Frequency of renewal: At the beginning and at the end of four intervals of 48 hours (nominal days 0 and 2, 6 and 8, 12 and 14, 18 and 20)
- Volume: 2 ml
- Storage: Samples were stored in a freezer until analysis.
At the end of the refreshment period, the replicates were pooled at each concentration before sampling.

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with a loading rate of 10 mg/l applying two days of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixture was left to stabilize for 1-1.5 hours where after the Water Accommodated Fraction (WAF) was siphoned off and used as the highest test concentration. All lower test concentrations were prepared by subsequent dilutions of the WAF in test medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.
Start of each batch : With newborn daphnids, i.e. less than 3 days old, by placing them individually in 50 ml M7-medium.
Maximum age of the cultures : 4 weeks
Temperature of medium : 18-22°C
Medium : M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
Light : 16 h photoperiod daily;
intensity at the start: 624-708 lux (8.8-10 μE.m-2.s-1)
intensity at the end: 612-710 lux (8.8-9.9 μE.m-2.s-1)
Feeding : Daily, 0.5 ml of Chlorella pyrenoidosa suspension containing 0.40-0.54 x108 cells/ml was added as feed for the daphnids. This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

Frequency of renewal: Every 48 hours

Post exposure observation period:

None

Hardness:

Total hardness varied between 179 and 214 mg calcium carbonate per litre, and thus complied to the requirements as laid down in the protocol (>140 mg CaCO3 per liter).

Test temperature:

The temperatures in the test media varied between 19.4 and 20.5°C. The temperature continuously measured in a temperature control vessel varied between 19.3 and 20.3°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

The pH remained within the range of 7.5 to 8.4 throughout the test and thus was maintained within the limits prescribed by the protocol (6.0-9.0, constant within 1.5 units).

Dissolved oxygen:

The oxygen concentration in all test solutions remained within the range of 8.2 to 9.7 mg/l during the exposure period and thus complied to the requirements as laid down in the protocol (>3 mg/l).

Salinity:

-

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32 and 100% of a WAF prepared at a loading rate of 10 mg/l
Measured concentrations: Analysis of the sample taken from the WAF prepared at 10 mg/l at the start of four renewal periods showed that the actual test concentration ranged between 4.2 and 6.6 mg/l. Test concentrations slightly decreased during the 48 hour renewal periods (to 70-82% of initial). Test concentrations containing 10 and 32% of the WAF proved to have been prepared properly and also slightly decreased during the 48 hour renewal periods (to 63-79% of initial. The test concentration containing 1.0% of the WAF was below the lowest calibration standard at all times, while the test concentration containing 3.2% of the WAF varied between being below or just above the lowest calibration solution. Given these results effect parameters were based on the average exposure concentrations: 0.040, 0.096, 0.42, 1.7 and 4.8 mg/l.

Details on test conditions:

Test duration: 21 days
Test type: Semi-static
Frequency of renewal: Every 48 hours
Test vessels: Volume: 60 ml (6 x Ø 3.5 cm), all-glass covered with a Perspex plate.
Medium: M7
Experimental design: At the start of the experiment (nominal day 0) 10 neonate daphnids, less than one day old, per group were divided over ten vessels each containing a minimum of 50 ml test medium. The control group consisted of 20 daphnids.
Light: 16 h photoperiod daily;
intensity at the start: 624-708 lux (8.8-10 µE.m-2.s-1)
intensity at the end: 612-710 lux (8.8-9.9 µE.m-2.s-1)
Feeding: Daily, 0.5 ml of Chlorella pyrenoidosa suspension containing 0.40-0.54 x10E8 cells/ml was added as feed for the daphnids. This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 4.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

(Parental mortality)

Remarks on result:

other: -

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

4.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

(Parental mortality)

Remarks on result:

other: -

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 0.91 - 2.2 mg/L

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: -

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: -

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 4.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Body length

Remarks on result:

other: -

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

4.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Body length

Remarks on result:

other: -

Details on results:

Condition of parental daphnids :
Three out of the twenty parental daphnids in the control died during the test period. Hence, parental mortality did not exceed 20% in the control. Mortality in all p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline concentrations tested was lower or slightly higher than in the control but not statistically significant (Fisher’s exact test, α=0.05).
In the control group, the presence of eggs in the brood pouch was recorded for the first time on day 4-6 and the first brood appeared between day 7 and day 9. The first recording of the presence of eggs in the brood pouch was similar to the control in treated solutions up to and including 4.8 mg/l. Hence, no delay in appearance of the first brood was observed in the treatments when compared to the control.

Reproduction:
The average cumulative number of young per female in the control group after 21 days was 136 with a coefficient of variation of 11%. The reproduction curves recorded at 0.040, 0.096 and 0.42 mg/l closely followed the curve of the control. The curve recorded at 1.7 mg/l stayed behind from the start and daphnids exposed to 4.8 mg/l did not reproduce at all. There were some recordings of immobile young and/or aborted eggs in the control and the three lowest test concentrations, while significant amounts of aborted eggs were recorded at the two highest test concentrations throughout the 21-day test period. Statistical analysis showed that the reproduction of the daphnids was significantly reduced at 1.7 mg/l and higher (Bonferroni t-Test, α=0.05).

Statistical analysis of the data on body length was not needed as the % reduction recorded was not biologically significant (<10%).

Results with reference substance (positive control):

-

Reported statistics and error estimates:

Statistical analysis:
Parental survival on day 21 was statistically tested using Fisher’s exact test (TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Total reproduction on day 21 was tested for normality and for homogeneity of variance and statistically tested using an ANOVA test followed by a mean comparison test (Bonferroni t-Test, TOXSTAT Release 3.5, 1996). The respective threshold levels of effect (LOEC) and the respective NOECs were determined on basis of these statistics.

EC-values:
The 21d-EC50 for reproduction was based on log-linear regression analysis of the percentages of reproductive reduction versus the logarithms of the corresponding average exposure concentrations of the test substance.

-

Validity criteria fulfilled:

yes

Conclusions:

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline did not affect reproduction of Daphnia magna at an average concentration of 0.42 mg/l after 21 days of exposure (NOEC).

Exposure to average concentrations of 1.7 mg/l and higher induced significant inhibition of the reproductive capacity of the parental daphnids (LOEC). The 21d-EC50 for reproduction was calculated to be 1.4 mg/l with a 95% confidence interval ranging from 0.91 to 2.2 mg/l.

Executive summary:

Exposure to average concentrations of 1.7 mg/l and higher induced significant inhibition of the reproductive capacity of the parental daphnids (LOEC). The 21d-EC50 for reproduction was calculated to be 1.4 mg/l with a 95% confidence interval ranging from 0.91 to 2.2 mg/l.

Also it was concluded that p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline did not affect reproduction of Daphnia magna at an average concentration of 0.42 mg/l after 21 days of exposure (NOEC).

Description of key information

The study performed to determine the long-term toxicity of the substance to aquatic invertebrate was a semi-static study on Daphnia magnia during 21 days according to OECD 211 and the Commission Regulation (EC) no 440/2008, C20. The study was testing the substance at the exposed concentrations of  0.040, 0.096, 0.42, 1.7 and 4.8 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.42 mg/L

Additional information

The study was testing the long term toxicity to aquatic invertebrates on Daphnia magnia under GLP conditions and according to OECD Guideline 211 "Daphnia magna, Reproduction Test" and was also meeting the test methods and validity criteria of the ISO International Standard 10706, 2000, the Commission Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

The exposure to average concentrations of 1.7 mg/l and higher induced significant inhibition of the reproductive capacity of the parental daphnids (LOEC for reproduction). The 21d-EC50 for reproduction was calculated to be 1.4 mg/l with a 95% confidence interval ranging from 0.91 to 2.2 mg/l.

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline did not affect reproduction of Daphnia magna at an average concentration of 0.42 mg/l after 21 days of exposure (NOEC for reproduction).