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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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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.