reaction product of toluenediisocyanate ( reaction mass of isomers: 65 % 2,4- and 35 % 2,6-diisocyanate) and aniline (molarratio 1:2)

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:

12 March 2018 - 16 May 2018

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:

October 02, 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 100 mg/L loading rate
- Sampling method: Duplicate samples were taken on days 0, 5, 12 and 19 (fresh test solutions) and 2, 7, 14 and 21 (old test solutions)
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (500 mg) was added to the surface of 5 litres of test water to give the 100 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixture allowed to stand for 1 hour. With the exception of the WAF preparation on Day 12 which showed no microscopic particles to be visible, microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the dispersed material by filtering the WAF through a glass wool plug (2 to 4 cm in length). On Day 12 the WAF was filtered through a glass wool plug (2 to 4 cm in length) as a precautionary measure. A wide bore glass tube, covered at one end with Nescofilm was submerged into the 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 Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed a clear colorless solution with no microdispersions of test item to be present.

Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. It was concluded that increasing the stirring period did increase the amount of dissolved test item in the WAF between the 24-hour and 96-hour preparation periods and so preparation of the WAF was maintained at a 95-hour stirring period followed by a 1-hour standing period.

- Controls: Untreated test media
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Microscopic observations of the WAF were performed after filtering and showed a clear colorless solution with no microdispersions of test item to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Not reported
- Source: In-house laboratory culture
- Age of parental stock (mean and range, SD): 1st instar < 24 hours old
- Feeding during test Yes
- Food type: Algal suspension and Tetramin® flake food suspension.
- Amount: Algal suspension (Desmodesmus subspicatus) and 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 animal.
- Frequency: Daily

ACCLIMATION
- Acclimation period: None

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

yes

Total exposure duration:

21 d

Post exposure observation period:

None

Hardness:

250 to 266 mg/L as CaCO3

Test temperature:

20 - 22 °C

pH:

7.7 - 8.4

Dissolved oxygen:

8.4 to 10.2 mg O2/L

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal loading rate: 100 mg/L
Measured:0.00729 to 0.0126 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel
- Type: closed
- Material, size, headspace, fill volume: 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Three times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 media prepared according to guideline
- Total organic carbon: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The temperature was also measured every hour in one replicate of the control. The water hardness of the control and the highest test concentration in the fresh and old media was measured once per week.

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 hours light and 8 hours darkness with 20 to 30 minute dawn and dusk transition periods
- Light intensity:880 - 1320 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. At the end of the test, the length of each surviving parent animal was determined

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable
- Range finding study: Yes
- Test concentrations: 1.0, 10 and 100 mg/L loading rates
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

not specified

Duration:

21 d

Dose descriptor:

EL50

Remarks:

Paternal generation

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Behavioural abnormalities: None reported
- Observations on body length and weight: Daily observations of the 100 mg/L test group recorded one Daphnia being observed as small, on five observations. After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P≥0.05) between the control and the 100 mg/L test group in terms of length of the daphnids after 21 days exposure to the test item.
- Other biological observations: Daily observations of the 100 mg/L test group recorded two observations of parental generation Daphnia being pale in coloration, and one observation of a Daphnia coated with algae.
- Mortality of control: 0
- Other adverse effects control: None reported
- Abnormal responses: None reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported; After siphoning and for the duration of the test, the control and 100 mg/L loading rate WAF test group were observed to be clear, colourless solutions.

Reported statistics and error estimates:

An estimate of the 21-Day EL50 value was given by inspection of the immobilization data. The EL50 (reproduction) value after 21 days was estimated by inspection of the data. For the estimation of the LOEL and the NOEL, the numbers of live young produced per adult over the duration of the test for the control and 100 mg/L test group were compared using a Two-sample t-test Procedure. Results from the control and 100 mg/L test group daphnia length data, determined for the surviving daphnids on termination of the test, were compared using a Two-sample t-test Procedure.

Table 1: Summary of the Lethal Effects on the Parental Generation (P₁)

Endpoint	Concentration (mg/L)	95 % Confidence Limits
Immobilisation EL10	> 100	Not determined
EL20	> 100	Not determined
EL50	> 100	Not determined
No Observed Effect Loading Rate	100	-
Lowest Observed Effect Loading Rate	> 100

Table 2: Summary of the Sub-lethal Effects on the Parental Generation (P₁)

Endpoint	Concentration (mg/L)	95 % Confidence Limits
Body Length EL10	> 100	Not determined
EL20	> 100	Not determined
EL50	> 100	Not determined
No Observed Effect Loading Rate	100	-
Lowest Observed Effect Loading Rate	>100

Table 3: Summary on the Effects on Reproduction

Endpoint	Concentration (mg/L)	95 % Confidence Limits
Body Length EL10	> 100	Not determined
EL20	> 100	Not determined
EL50	> 100	Not determined
No Observed Effect Loading Rate	100	-
Lowest Observed Effect Loading Rate	> 100	-

Table 4: Range-finding test data

Nominal Loading Rate (mg/L)	Percentage Immobilization After 10 days exposure	Sub-lethal effect	Average Number of Live Young in First brood
Control	0	None	13.6
1.0	40	Pale	8.0
10	60	Pale	7.0
100	80	Pale	8.0

Nominal Loading Rate (mg/L)	Percentage Immobilization After 10 days exposure	Sub-lethal effect	Average Number of Live Young in First brood
Control	100	None	0.0*^ǂ
1.0	0	Small	11.0*
10	0	Pale	10.2
100	0	Small & Pale	13.6

*Based on four replactes as one replicate recorded as being male

ǂ All parental daphnia died by Day 4, hence no offspring produced

Table 5: Summary of Findings Following the Exposure of Daphnia magna for 21 days

Nominal Loading Rate    (mg/L) WAF	Parental (P1 Generation Mortalities       (Initial Population = 10)			Total Number of Live Young	Number of Live Young from Parents Surviving to the End of the Test		Number of Live Young at the End of the Test Excluding Parents that were Accidental or Inadvertent Mortalities
	Total	Accidental	Inadvertant		Total	Average per Parent	Total	Average Per Parent
Control	0	0	0	1734	1734	173	1734	173
100	1	0	1	1592	1580	176	1580	176

 

Table 6: Summary of Daily Observations

Day	Nominal Loading Rate Concentration
	Control		100
	Adults Surviving	Live Young	Adults Surviving	Live Young
1	10	0	10	0
2	10	0	10	0
3	10	0	10	0
4	10	0	10	0
5	10	0	10	0
6	10	0	10	0
7	10	0	10	0
8	10	0	10	0
9	10	0	9	25
10	10	159	9	127
11	10	0	9	0
12	10	24	9	103
13	10	242	9	142
14	10	0	9	0
15	10	367	9	376
16	10	91	9	38
17	10	0	9	0
18	10	325	9	306
19	10	191	9	87
20	10	0	9	0
21	10	335	9	388
Totals	10	1734	9	1592

Table 7: Total Cumulative Production of Live Young

Nominal Loading Rate (mg/L)	Day
	1	2	3	4	5	6	7	8	9	10
Control	0	0	0	0	0	0	0	0	0	159
100	0	0	0	0	0	0	0	0	25	152

Nominal Loading Rate (mg/L)	Day
	11	12	13	14	15	16	17	18	19	20	21
Control	159	183	425	425	792	883	883	1208	1399	1399	1734
100	152	255	397	397	773	811	811	1117	1204	1204	1592

Table 8: Body Length of Surviving Adults at Day 21

Nominal Loading Rate (mg/L)	Individual Daphnia Lengths (mm)
	1	2	3	4	5	6	7	8	9	10	Av	SD
Control	4.4	4.5	4.1	4.3	4.7	4.4	4.5	4.8	4.7	4.5	4.5	0.21
100	4.4	3.9	4.5	4.2	-	4.5	4.5	4.4	4.3	4.9	4.4	0.27

Av = Average

SD = Standard Deviation

- = No measurement, Daphnia died during the test

Table 9: Water Quality Measurements

Day	Control			100 mg/L
	mg O2/L	pH	Temperature (°C)	mg O2/L	pH	Temperature (°C)
0	8.7	7.9	21	8.6	7.8	22
2*	9.1	7.7	20	9.2	7.9	21
2^+	8.7	7.9	21	8.5	7.9	22
5*	9.4	8.1	20	10.1	8.4	20
5^+	9.2	8.0	21	8.9	8.2	21
7*	9.1	8.1	20	9.0	8.1	21
7^+	8.8	8.1	20	8.8	8.1	21
9*	9.1	8.2	20	9.1	8.1	20
9^+	9.0	7.9	21	8.9	7.9	21
12*	9.2	8.2	21	9.0	8.1	21
12^+	9.0	8.1	21	8.9	8.1	21
14*	9.1	8.1	21	9.0	8.1	21
14^+	8.7	8.0	22	8.4	8.1	22
16 *	8.9	8.1	21	8.5	8.1	21
16^+	9.2	8.2	20	9.1	8.2	21
19 *	10.2	8.1	20	9.9	8.2	20
19^+	8.9	8.1	21	8.9	8.2	21
21	9.9	8.1	20	9.5	8.1	20

*Before renewal

⁺After renewal

Table 10: Water Hardness Measurements

Day	Water Hardness (mg/L as CaCO3)
	Control	Highest Surviving Test Conditions
0 (Fresh media)	256	258
2 (Old media)	262	258
5 (Fresh media)	250	256
7 (Old media)	252	260
12 (Fresh media)	260	260
14 (Old media)	264	264
19 (Fresh media)	256	258
21 (Old media)	262	266

Table 11: Light Intensity Measurements

Day	Light Intensity (Lux)
0	880
1	890
2	929
3	980
4	975
5	946
6	1167
7	1142
8	1320
9	1159
10	1151
11	928
12	1017
13	1068
14	996
15	1069
16	1078
17	1108
18	1163
19	1289
20	1346
21	1068

Table 12: Concentrations in test samples

Time point (Days)	Nominal Loading Rate [Cnom] (mg/L WAF)	Sample Preparation Factor [F]	Determined Concentration [c] (mg/L)
0 (fresh)	Control	0.2	<LOQ
	100	0.4	0.00929
2 (old)	Control	0.2	<LOQ
	100	0.2	0.0103
5 (fresh)	Control	0.2	<LOQ
	100	0.4	0.0126
7 (old)	Control	0.2	<LOQ
	100	0.2	0.0111
12 (fresh)	Control	0.2	<LOQ
	100	0.4	0.00909
14 (old)	Control	0.2	<LOQ
	100	0.2	0.00932
19 (fresh)	Control	0.2	<LOQ
	100	0.4	0.00729
21 (old)	Control	0.2	<LOQ
	100	0.2	0.00874

LOQ = Limit of Quantification

Validity criteria fulfilled:

yes

Conclusions:

The 21-Day EL50 (immobilization) value for the parental daphnia generation (P1) and the 21-Day EL50 (reproduction) were >100 mg/L, based on nominal loading rates. The No Observed Effect Loading Rate based on nominal loading rates was 100 mg/L, the only concentration tested.

Executive summary:

The long-term toxicity of the test item to Daphnia magna was determined in a study following OECD 211 and EC C.20 guidelines. The study was conducted as a limit test at a nominal loading rate of 100 mg test item/L, prepared as a water accommodated fraction, based on the results of a preliminary study. Test solutions were renewed 3 times per week. Test concentrations were analysed and determined to be between 0.00729 mg/L and 0.0126 mg/L over the course of the study. 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. At the end of the test, the length of each surviving parent animal was determined. The 21-Day EL50 (immobilization) value for the parental daphnia generation (P1) and the 21-Day EL50 (reproduction) were >100 mg/L, based on nominal loading rates. The No Observed Effect Loading Rate based on nominal loading rates was 100 mg/L, the only concentration tested. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test substance as a whole, the results are reported based on nominal loading rates only.

 

The study is a GLP compliant, guideline experimental study and is fully acceptable for assessment of this endpoint.

Description of key information

The long-term toxicity to Daphnia magna has been assessed for one substance within the TDI-II category definition. The study was conducted using a water accommodated fraction as the test item is a poorly soluble UVCB. The determined EL50 values, based on parental mortality and reproduction, were >100 mg/L, with a NOELR value of 100 mg/L, the only concentration tested. Based on these results, members of the TDI-II category are not expected to be toxic to aquatic invertebrates at the limit of solubility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

A study was performed to assess the long-term toxicity of the Reaction mass of aniline and m-tolylidene diisocyanate to Daphnia magna (Sacker 2018). The results from this study are read across to other members of the polyurea TDI-II category.

The method followed was designed to be compatible with the OECD guideline 211, "Daphnia magna, Reproduction Test". Following preliminary range-finding tests, ten daphnids (ten replicates of one animal) were exposed to a Water Accommodated Fraction (WAF), at a single nominal loading rate of 100 mg/L, with test media renewal three times per week. The stirring period for the WAF preparation was validated in a preliminary study before the main test. The study was conducted with 21 days exposure. Test concentrations were analysed and determined to be between 0.00729 mg/L and 0.0126 mg/L over the course of the study. 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. At the end of the test, the length of each surviving parent animal was determined.

The 21-Day EL50 (immobilization) value for the parental daphnia generation (P1) and the 21-Day EL50 (reproduction) were >100 mg/L, based on nominal loading rates. The No Observed Effect Loading Rate based on nominal loading rates was 100 mg/L, the only concentration tested. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test substance as a whole, the results were based on nominal loading rates only.