Reaction mass of 4,4'-methylenediphenyl diisocyanate and Amines, soya alkyl

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:

07 December 2017 to 14 March 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
- 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 (400 mg) was added to the surface of 4 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 23 hours and the mixture allowed to stand for 1-Hour. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 100 mg/L loading rate WAF.

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 not 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 23-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 inspection of the WAF showed no micro-dispersions or undissolved 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: 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, with the exception of Day 0 when the concentrated algal suspension was used in error. Equal amounts of food were given to each daphnid.
- 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:

228 to 260 mg/L as CaCO3

Test temperature:

19 - 21 °C

pH:

7.7 to 8.4

Dissolved oxygen:

7.8 - 9.5 mg/L

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal loading rate: 100 mg/L
Measured: < LOQ

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, with the exception of the first week when no old sample was taken in error.

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 674 - 952 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: 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: None reported
- 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: Range-finding Test data

Nominal Loading Rate (mg/L)	Percentage Immobilisation After 10 Days Exposure	Sub-lethal Effects	Total Number of Live Young
Control	0	None	129
1.0	0	None	126
10	0	None	134
100	0	None	132

Table 2: Summary of finding following the expousre of Daphnia magna for 21 days

Nominal Loading Rate (mg/L)	Parental (P1) Generation Motalities (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	1730	1730	173	1730	173
100	0	0	0	1626	1626	163	1626	163

Table 3: Summary of Observations in the Definitive Test

Day	Nominal Loading Rate
	Control		100 mg/L
	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	157	10	145
9	10	0	10	0
10	10	0	10	0
11	10	300	10	298
12	10	0	10	0
13	10	0	10	115
14	10	432	10	286
15	10	0	10	0
16	10	0	10	0
17	10	457	10	441
18	10	0	10	0
19	10	0	10	0
20	8	384	10	341
21	8	0	10	0
TOTALS	8	1730	10	1626

Table 4: Total cumulative production of Live Young

Nominal Loading Rate (mg/L)	Day
	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Control	0	0	0	0	0	0	0	157	157	157	457	457	457	889	889	889	1346	1346	1346	1730	1730
100	0	0	0	0	0	0	0	145	145	145	443	443	558	844	844	844	1285	1285	1285	1626	1626

Table 5: 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
Control	4.5	4.5	4.5	4.4	4.5	4.4	4.4	4.5	4.4	4.4
100	4.5	4.3	4.4	4.4	4.5	4.5	4.6	4.3	4.1	4.4

Table 6: Water Quality Measurements

Nominal Loading Rate (mg/L)		Day
		0	1	2*	2+	3	4*	4+	5	6*	6+	7	8	8	9	10
Control	mg O2/L	8.7	-	9.4	8.9	-	9.0	8.8	-	9.2	8.9	-	9.2	8.9	-	-
	pH	8.0	-	8.4	8.1	-	8.1	7.9	-	8.0	8.0	-	8.0	8.0	-	-
	Temperature (°C)	21	20	20	21	20	21	21	21	20	21	20	20	20	20	20
100	mg O2/L	8.7	-	9.2	8.7	-	9.0	8.7	-	9.2	8.9	-	9.3	8.9	-	-
	pH	8.1	-	8.3	8.1	-	8.1	8.0	-	8.1	8.0	-	8.0	8.0	-	-
	Temperature (°C)	21	20	20	21	20	21	21	20	20	21	20	20	21	20	20

Nominal Loading Rate (mg/L)		Day
		11*	11+	12	13*	13+	14	15*	15+	16	17	18*	18+	19	20*	20+	21
Control	mg O2/L	9.5	9.0	-	8.9	9.0	-	8.4	8.7	-	-	8.4	8.5	-	7.8	8.8	8.5
	pH	8.2	8.0	-	8.0	7.9	-	7.8	7.7	-	-	7.9	8.0	-	7.8	7.8	8.0
	Temperature (°C)	19	20	20	20	20	19	20	20	20	20	20	21	20	20	21	20
100	mg O2/L	9.4	8.9	-	9.0	8.9	-	8.5	8.7	-	-	8.4	8.5	-	8.0	8.6	8.4
	pH	8.0	8.0	-	8.1	8.0	-	7.8	7.9	-	-	7.9	8.0	-	7.8	7.9	7.9
	Temperature (°C)	19	20	20	20	21	19	20	21	20	20	20	21	20	20	21	20

* Before renewal

+ After renewal

- Not required

Table 7: Results for Test samples

Time Point (Day)	Nominal Loading Rate cnom (mg/L)	Sample Prepartion Factor F	Determined Concentration of Test Item in Test sample c (mg/L)
0	Control	0.1	<LOQ
Fresh	100	0.1	<LOQ
4	Control	0.1	<LOQ
Fresh	100	0.1	<LOQ
6	Control	0.1	<LOQ
Old	100	0.1	<LOQ
11	Control	0.1	<LOQ
Fresh	100	0.1	<LOQ
13	Control	0.1	<LOQ
Old	100	0.1	<LOQ
20	Control	0.1	<LOQ
Fresh	100	0.1	<LOQ
21	Control	0.1	<LOQ
Old	100	0.1	<LOQ

Validity criteria fulfilled:

yes

Remarks:

Control mortality 0%; Mean number of live young (control): 173; coefficient of variation (control): 7%; No ephippia produced: 0; dissolved oxygen: >=7.8 mg/L; pH (control): 7.7 - 8.4 (variation 0.7)

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. 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 substances as a whole, and the dissolved concentrations were below the quantifiable limit of the analytical method, 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 MDI 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 MDI 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 product of 4,4'-methylenediphenyl diisocyanate and amines, coco alkyl to Daphnia magna (Ablitt 2018). The results from this study are read across to other members of the polyurea MDI 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 at a temperature of 19°C to 21°C. Chemical analysis of the freshly prepared test preparations on Days 0, 4, 11 and 20, and of the old or expired media on Days 6, 13 and 21 was conducted and showed measured test concentrations of less than the LOQ of the analytical method employed were obtained which was determined to be 0.00060 mg/L. 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, and the dissolved concentrations were below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.