Reaction products of 1,4-cyclohexanedimethanol with propylene oxide and ammonia

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:

Experimental start date 24 August 2016 and Experimental completion date 22 December 2016

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:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

By high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard.

Details on sampling:

- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give test solutions of 0.625, 1.25, 2.5, 5.0, 10 and 20 mg/L.
The stock solution and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh test solutions on Days 0, 5, 14 and 19, and in the expired test solutions on Days 2, 7, 16 and 21.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Annex 2) in a temperature controlled room at approximately 20 °C. 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 mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

248 to 270 mg/L as CaCO3

Test temperature:

19-22°C

pH:

7.2-8.5

Dissolved oxygen:

8.6 to 9.7 mg O2/L

Salinity:

not applicable as fresh water used for the experiment

Nominal and measured concentrations:

Nominal concentrations: 0.625, 1.25, 2.5, 5.0, 10.0 and 20.0 mg/L
Measured concentrations: Analysis of the fresh test preparations on Days 0, 5, 14 and 19 showed measured test concentrations to range from 96% to 122% of the nominal test concentrations. There was no significant change in the measured concentrations of the aged test preparations on Days 2, 7, 16 and 21, and so the results are based on the nominal test concentrations only.

Details on test conditions:

Exposure Conditions
TEST SYSTEM:
For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels, which were then covered with a plastic lid to reduce evaporation. For each test and control group, ten replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions, but not exposed to the test item.
The test preparations were renewed 3 times per week on Days 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 discarded or unhatched eggs were collected on the mesh, and counted before being discarded.
Each daphnid received approximately 5 to 15 μL of an algal suspension (Desmodesmus subspicatus) and approximately 20 μL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minutes dawn and dusk transition periods for 10 days.
- Light intensity: 385 to 581 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 or unhatched eggs were counted. An assessment was also made of the general condition and size of the parental daphnia, and the filial daphnia when present, as compared with the controls.

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 (ie. immobile) were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of offspring produced in the test vessels.

At the end of the test, the length of each surviving parent animal was determined.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- In range finding test factor: 10
- In Definitive test factor : 2

- Range finding study
- Test concentrations: 0.1, 1.0, 10.0 and 100.0 mg/L

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

8.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Range-finding Test:
No immobilization in the adult daphnia was observed at the test concentrations of 0.10 and 1.0 mg/L, however, immobilization was observed in the 10 and 100 mg/L.
Sub-lethal effects of exposure were observed in the 10 mg/L test concentration in the adult daphnia (pale color, small size).
Chemical analysis of the 1.0 and 10 mg/L test preparations on Day 0 (see Annex 3) showed measured concentrations of 1.03 and 9.71 mg/L, respectively. There was no significant change in the measured concentrations of the aged test preparations on Day 2 indicating that the test item was stable under test conditions.

Definitive Test
Based on the results of the preliminary range-finding test, test concentrations of 0.625, 1.25, 2.5, 5.0, 10 and 20 mg/L were selected for the definitive test.

Verification of Test Concentrations
Analysis of the fresh test preparations on Days 0, 5, 14 and 19 showed measured test concentrations to range from 96% to 122% of the nominal test concentrations (see Annex 3). There was no significant change in the measured concentrations of the aged test preparations on Days 2, 7, 16 and 21, and so the results are based on the nominal test concentrations only.
There were three measured concentrations that were greater than 80% to 120% of the nominal test concentrations (121% and 122% of nominal). These were considered slight differences, and therefore, all measured concentrations were considered acceptable.

Validation Criteria
Please see below.

Lethal Effects on the Parental Generation (P1)
There was a single parental daphnid mortality observed in the 1.25 and 10 mg/L test concentrations on Day 1. There was no other mortality in the 0.625 to 5.0 mg/L test groups for the duration of the test, while further mortalities occurred in the 10 mg/L test group starting on Day 7. Based on the timing of these initial mortalities (prior to Day 7) they were considered to be due to accidental/inadvertent causes and were removed from analysis.
The 10 mg/L test group had 56% immobilization with mortalities occurring from Days 7 to 19. The 20 mg/L test concentration had 100% immobilization by Day 6. The mortalities were considered to be due to toxic effect attributable to exposure of Daphnia magna to the test item.
The following ECx (immobilization) values based on nominal test concentrations were calculated by the Maximum-Likelihood Probit method at 21 days:

EC10 = 8.2 mg/L (immobilization) and 95% confidence limits was not determined.
EC20 = 8.7 mg/L (immobilization) and 95% confidence limits was not determined.
EC50 = 9.8 mg/L (immobilization) and 95% confidence limits was not determined.

Sub-lethal Effects on the Parental Generation (P1)
Replicate 4 of the 1.25 mg/L test group was observed to be pale in color from Day 13 to 21.
In the 10 mg/L test concentration, there were significant sub-lethal effects on the parental daphnids throughout the study. Replicate 1 was observed to be pale in color on Days 20 and 21. Replicate 2 was observed to be pale on Days 4 to 6. Replicate 3 was observed to be pale on Days 14 to 18. Replicate 4 was observed to be pale on Days 16 to 21. Replicate 5 was observed to be pale on Days 20 and 21. Replicate 6 was observed to be pale and small in size on Days 12 to 17. Replicate 7 was observed to be pale on Days 5 to 6, pale and small on Days 7 and 10, and small on Days 8 to 9. Replicate 8 was observed to be pale on Days 16, 17, 19, 20 and 21. Replicate 9 was observed to be pale on Days 4 to 6, pale and small on Day 7, and pale, small and have reduced mobility on Day 8.
In the 20 mg/L test concentration, the surviving parental daphnids were observed to be pale in color from Day 2 to 4, and pale with reduced mobility on Day 5.
After 21 days, the length of each surviving adult was determined, the results of which are given in Table 5. The results showed that there were no statistically significant differences (P≥ 0.05) between the control and the 0.625, 1.25, 2.5, 5.0 and 10 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item (see Appendix 1). The 20 mg/L test group data was not included in this analysis as exposure to the test item eliminated all the daphnids prior to Day 21 of the test.

Effects on Reproduction
After 21 days there were no statistically significant differences between the control and the 0.625, 1.25, 2.5, 5.0 and 10 mg/L test groups in terms of the number of live young produced per adult (see Appendix 1).
The following ECx (reproduction) values based on nominal test concentrations were calculated by the Maximum-Likelihood Probit method at 21 days:
EC10 = 9.1 mg/L (reproduction) and 95% confidence limits was not determined.
EC20 = 9.4 mg/L (reproduction) and 95% confidence limits was not determined.
EC50 = 10 mg/L (reproduction) and 95% confidence limits was not determined.

Effects on the Filial Generation (F1)
Information on the effects of the test item 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 the 0.625 mg/L test group were in the same general condition as the young produced by the control over the duration of the test. 3
The young produced by the 1.25 to 10 mg/L test groups were observed to be similar when compared to the control young except as noted.
In the 1.25 mg/L test group, a single brood produced on Day 14 was observed to be pale and small. On Day 20, 3 broods were observed to be pale in color.
In the 2.5 mg/L test group, a single brood produced on Days 12 and 20 were observed to be pale in color. On Day 20, a single brood was observed to be small.
In the 5.0 mg/L test group, all broods produced on Day 13 and the majority of broods on Day 16 were observed to be pale in color. On Day 16, a single brood was observed to be small.
In the 10 mg/L test group, the majority of broods produced from Days 12 to 21 were observed to be pale, small or both.
Exposure to the test item eliminated the parental generation (P1) of the 20 mg/L test group prior to the production of young.
Young were first produced in the control test group on Day 9 of the test.
There were no unhatched eggs or dead young observed in all control and treatment groups surviving to maturation.


Lowest Observed Effect Concentration
The "Lowest Observed Effect Concentration" (LOEC) for reproduction was 20 mg/L as this test group produced significantly fewer live young per adult (p < 0.05) than the control group.
The "Lowest Observed Effect Concentration" (LOEC) for mortality (immobilization) was 10 mg/L as this test group had significantly higher mortalities (immobilization) (p < 0.05) in the parental generation (P1) when compared to the control after 21 days.


No Observed Effect Concentration
The "No Observed Effect Concentration" (NOEC) for reproduction was 10 mg/L as there were no significant differences (p ≥ 0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.
The "No Observed Effect Concentration" (NOEC) for mortality (immobilization) was 5.0 mg/L as there were no significant mortalities (p ≥ 0.05) observed in the parental generation (P1) when compared to the control after 21 days


Analysis of the 0.625 to 10 mg/L Test Groups
Due to the elimination of the 20 mg/L parental generation (P1) prior to any reproduction, the Sponsor requested a separate statistical analysis of only the 0.625 mg/L to 10 mg/L test group.

Water Quality Criteria
Temperature was maintained at 19 °C to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The water hardness was observed to be in the range 248 to 270 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

CONCLUSION
Exposure of Daphnia magna to the test item resulted in significant mortalities at the concentrations of 10 and 20 mg/L resulting in 60% and 100% mortalities by Days 19 and 6, respectively.
The 21-Day EC50 (immobilization) value, based on nominal concentrations, for the parental daphnia generation (P1) was calculated to be 9.8 mg/L.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) for immobilization based on nominal concentrations were 10 and 5.0 mg/L, respectively.
No significant impairment of reproduction was observed at the 0.625, 1.25, 2.5, 5.0 and 10 mg/L test concentrations during the test.
The 21-Day EC50 (reproduction) based on nominal concentrations was 10 mg/L.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) for reproduction based on nominal concentrations were 20 and 10 mg/L, respectively.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis:
Please see "Any other infromation on results incl tables" field.

Validation Criteria

The following validation criteria were achieved during the test.

	Required	Actual
a) Control mortality	≤ 20%	0%
b) Mean number of live young per surviving adult (control group)	≥ 60 after 21 days	152
c) Coefficient of variation for control group ∗	≤ 25%	7.2%
d) No ephippia produced	0	0
e) Dissolved oxygen	> 3 mg O2/L	≥ 8.0 mg O2/L
f) pH (control group)	6 to 9	7.2 to 8.0
	Variation ≤ 1.5	0.8

Statistical Analysis

Analysis of Numbers of Live Young Produced per Adult

Results from the control and each test group were compared using Levene’s test on variance homogeneity and the Multiple sequentially-rejective Median (2x2-table) Test after Bonferroni-Holm.

Mean and standard deviation values derived from the number of live young produced per introduced adult in the control and the test groups, excluding any parent daphnia which may have accidentally or inadvertently died during the test, were used for this analysis and are given in the table below:

 

Nominal Test Concentration (mg/L)		Number of Live Young Produced per Adult by Day 21
Control	Mean	152
Control	Standard Deviation	11
0.625	Mean	151
0.625	Standard Deviation	9
1.25	Mean	153
1.25	Standard Deviation	38
2.5	Mean	180
2.5	Standard Deviation	8
5.0	Mean	171
5.0	Standard Deviation	15
10	Mean	78
10	Standard Deviation	93
20	Mean	0
20	Standard Deviation	0

No significant differences (p ≥ 0.05) were found between the control and the 0.625 to 10 mg/L test groups in terms of the number of live young produced per adult by Day 21 using the above methods of statistical analysis. However, there was a significant difference found between the control and 20 mg/L test group.

Analysis of the Daphnia Length Data

Results from the control and the 0.625 mg/L to 10 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 Levene’s test for homogeneity of variance and Dunnett’s multiple t-test procedure for comparing several treatments with a control.

Mean and standard deviation values derived from the length of the surviving daphnids of the parental (P1) generation in the control and the 0.625 mg/L to 10 mg/L test groups were used for this analysis and are given in the table below:

Nominal Test Concentration (mg/L)		Daphnia Lengths at Day 21 (mm)
Control	Mean	4.0
Control	Standard Deviation	0.22
0.625	Mean	4.1
0.625	Standard Deviation	0.21
1.25	Mean	4.0
1.25	Standard Deviation	0.20
2.5	Mean	4.1
2.5	Standard Deviation	0.25
5.0	Mean	4.3
5.0	Standard Deviation	0.30
10	Mean	4.0
10	Standard Deviation	0.31

No statistically significant differences (p ≥ 0.05) were found between the control and the 0.625 mg/L to 10 mg/L test groups in terms of length of the surviving parental daphnids on Day 21 of the test, using the above methods of statistical analyses. The 20 mg/L test group was omitted from the analyses as there were no surviving daphnids on Day 21.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in significant mortalities at the concentrations of 10 and 20 mg/L resulting in 60% and 100% mortalities by Days 19 and 6, respectively.
The 21-Day EC50 (immobilization) value, based on nominal concentrations, for the parental daphnia generation (P1) was calculated to be 9.8 mg/L.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) for immobilization based on nominal concentrations were 10 and 5.0 mg/L, respectively.
No significant impairment of reproduction was observed at the 0.625, 1.25, 2.5, 5.0 and 10 mg/L test concentrations during the test.
The 21-Day EC50 (reproduction) based on nominal concentrations was 10 mg/L.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) for reproduction based on nominal concentrations were 20 and 10 mg/L, respectively.

Executive summary:

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No. 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.625, 1.25, 2.5, 5.0, 10 and 20 mg/L for a period of 21 days. The test solutions were renewed 3 times per week throughout the test.

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.

Analysis of the fresh test preparations on Days 0, 5, 14 and 19 showed measured test concentrations to range from 96% to 122% of the nominal test concentrations. There was no significant change in the measured concentrations of the aged test preparations on Days 2, 7, 16 and 21, and so the results are based on the nominal test concentrations only.

There were three measured concentrations that were greater than 80% to 120% of the nominal test concentrations (121% and 122% of nominal). These were considered slight differences, and therefore, all measured concentrations were considered acceptable.

Exposure of Daphnia magna to the test item gave the following results based on the nominal test concentrations:

Endpoint		Concentration (mg/L)
Immobilization	EC10 95% Confidence limits	8.2 Not determined
Immobilization	EC20 95% Confidence limits	8.7 Not determined
Immobilization	EC50 95% Confidence limits	9.8 Not determined
No Observed Effect Concentration (NOEC)		5.0
Lowest Observed Effect Concentration (LOEC)		10

Endpoint		Concentration (mg/L)
Reproduction	EC10 95% Confidence limits	9.1 Not determined
Reproduction	EC20 95% Confidence limits	9.4 Not determined
Reproduction	EC50 95% Confidence limits	10 Not determined
No Observed Effect Concentration (NOEC)		10
Lowest Observed Effect Concentration (LOEC)		20

Description of key information

In this study, the chronic toxicity of Jeffamine RFD 270 to Daphnia magna was investigated in a 21 -day study according to OECD 211 with concentrations between 0.625 and 20mg/L, resulting in an EC10 of 9.1 mg/L for reproduction and in an EC10 of 8.2 mg/L for immobilisation.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.2 mg/L