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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:
12 Jun 2018 - 03 Sep 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:
2012
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
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and the control
- Sampling method: 2.0 mL, At the beginning and at the end of three intervals of 48 hours (nominal days 0 and 2, 8 and 10, 16 and 18)
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility
- At the end of the refreshment period, the replicates were pooled at each concentration before sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions for the reproduction test started with the highest concentration of 10 mg solids/L (26 mg test item/L) applying an one-hour period of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives
- Other relevant information: Test item was completely soluble in test medium at the concentrations tested in the reproduction test. A correction was made for the purity/composition of the test substance. A correction factor of 2.554 was used. Concentration/doses are expressed as solids.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna Straus 1820
- Age at study initiation: <24 h old, from parental daphnids > 2 weeks old
- Method of breeding: cyclical parthenogenesis under specified breeding conditions
- Source: In-house laboratory culture with a known history.
- Feeding during test : Twice daily an amount of 0.25 ml of a Chlorella sp. suspension (corresponding to 0.2 mg C/Daphnia/day). On weekend days an amount of 0.50 ml was added in one single feed.
- 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.

CULTIVATION
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
161 - 179 mg/L as CaCO3
Test temperature:
19 - 21 °C
pH:
7.6 - 8.6
Dissolved oxygen:
6.9 - 9.7 mg O2/L
Nominal and measured concentrations:
Nominal: 0.46, 1.0, 2.2, 4.6, 10 mg solids/L, corresponding to 1.2, 2.6, 5.6, 12 and 26 mg/L.
Measured concentrations: Concentrations were unstable during the refreshment periods. Time Weight Mean concentrations were calculated: 0.075, 0.15, 0.45, 1.6 and 3.7 mg solids/L. See 'Any other information on materials and methods' for calculation of TWM, see 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass vessel, covered with a Perspex plate. Volume: 60 mL (6 x Ø 3.5 cm), fill volume 50 mL
- Aeration: no
- Renewal of test medium: every 48 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7 medium according to OECD 211
- Total organic carbon: < 2 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature, oxygen and pH: At the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids. Hardness: Once a week in fresh and old media from the control and the highest test concentration. Light: At the start and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h photoperiod daily
- Light intensity: 517-607 Lux (start test), 492-607 Lux (end test).

EFFECT PARAMETERS MEASURED
- Parental daphnids:
* Condition: Every workday and upon renewal on non-workdays, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
* Presence of eggs in the brood pouch: Every workday and upon renewal on non-workdays.
* Body length: At the end of the test.
- Offspring
* Appearance first brood: When observed.
* Newborn daphnids: Every workday and upon renewal on non-workdays, the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
* Presence of unhatched eggs: When observed.
* Incidence of immobility: When observed.

TEST CONCENTRATIONS
- Range finding study test concentrations: 0, 1.0, 10, 100 mg solids/L (Water Accommodated Fractions)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
reproduction
Details on results:
Analytical results:
Concentrations measured in freshly prepared solutions ranged from 47 to 133% of nominal concentrations, with majority of results being within 87-120% of nominal. The concentration of one sample (2.2 mg solids/L at day 16) was found to be 47% of nominal.
Concentrations measured in spent solutions at the end of the first refreshment interval were at the level of 0.11 to 78% of initially measured. Stability of test concentrations decreased with decreasing nominal concentration. Such relation was not observed for the two other refreshments. In these two renewals, significantly stronger decrease of concentrations was observed than in case of the first renewal. Hence, the stability of the test solutions during the renewal periods decreased as exposure progressed. This decrease was suspected to be related to consumption and or absorption of test item by the parental daphnids and the increasing numbers of young towards the end of the 21-day exposure period.

Condition of parental daphnids:
Four out of the twenty parental daphnids died during the test period in the control treatment. Hence, parental mortality did not exceed 20% in the controls. Mortality observed in the test item treated groups ranged from 10 to 40%. Statistical analysis indicated that there was no significant trend in mortality of parental daphnids, hence the effects on reproduction were expressed in terms of number of offspring per surviving parental daphnid.

Time to first reproduction:
First offspring were observed on day 7 in all groups but the highest one. In the highest concentration, first offspring were observed on day 8 of exposure.

Reproduction:
On average, 198 offspring were produced per surviving daphnid in the control treatment. The coefficient of variation for the reproduction in the control was 5.7%. An increase of reproduction, rather than a reduction was observed in all groups tested. There were no recordings of immobile young or appearance of unhatched (aborted) eggs at any of the test concentrations throughout the 21-day test period

Body length:
Change in the mean body length ranged between -1.0 to 1.8% was observed at the four lowest concentrations and was not dose-related. At the highest concentration, a reduction of 4.6% was observed. This reduction was statistically significant

Experimental conditions:
All environmental parameters remained within the requirements as laid down in the study plan.

Effect parameters:
NOEC for reproduction: 3.7
EC10 for reproduction: >3.7
EC50 for reproduction: >3.7
NOEC for mortality: 3.7
NOEC for growth reduction: 1.6
EC10 for growth reduction: >3.7
Reported statistics and error estimates:
Mortality of parental daphnids was analysed with Fisher`s exact binomial test with Bonferroni correction (α=0.05, one-sided greater) after qualitative trend analysis by contrasts (monotonicity of concentration/response).

NOEC for growth was determined as follows: Data distribution: Shapiro-Wilk's Test; Homogeneity of variance: Levene's Test (with Residuals); Differences between treatments and the control: Williams multiple sequential t-test procedure (α=0.05, one-sided smaller) after trend analysis by contrasts (monotonicity of concentration/response).

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Nominal and Mean Measured Exposure Concentrations

Nominal

concentration

(mg solids/L)

Measured concentration (mg solids/L) at a given day

Time

Weighted

Mean

(mg solids/L)

0

2

8

10

16

18

Fresh

Old

Fresh

Old

Fresh

Old

0.46

0.45

0.00052

0.48

0.0022

0.51

0.00033

0.075

1.0

0.78

0.00090

1.2

0.00045

1.0

0.0059

0.15

2.2

2.1

0.28

2.4

0.00096

1.0

0.00040

0.45

4.6

5.2

2.5

6.1

0.00089

4.7

0.00043

1.6

10

8.7

6.8

10

0.12

12

0.00053

3.7

 

Table 2: Mortality (Immobility) of Parental Daphnids at the End of the Test

Test item

TWM concentration (mg solids/L)

Introduced

Mobile

Immobile

% Immobility

Control

20

16

4

20

0.075

10

8

2

20

0.15

10

8

2

20

0.45

10

9

1

10

1.6

10

7

3

30

3.7

10

6

4

40

Table 3: Group Mean Cumulative Number of Juveniles per Surviving Parent and Reduction of Reproduction at the End of the Test 

Test item

TWM concentration (mg solids/L)

Mean

Std. Dev.

n

%Reduction

Control

198.3

11.37

16

0.075

202.4

17.93

8

-2.0

0.15

214.5

34.64

8

-8.2

0.45

227.9

16.56

9

-14.9

1.6

235.9

13.56

7

-18.9

3.7

199.2

9.60

6

-0.4

Table 4: Group Mean Body Lengths (mm) and Reduction of Growth of Parental Daphnids at the End of the Test

Test item

TWM concentration (mg solids/L)

Mean

(mm)

Std. Dev.

n

%Reduction

Control

4.59

0.159

16

 

0.075

4.52

0.128

8

1.6

0.15

4.64

0.110

8

-1.0

0.45

4.51

0.156

9

1.8

1.6

4.60

0.235

7

-0.1

3.7

4.38

0.136

6

4.6*

*  Statistically significant (p<0.05)

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The test item did not exert any inhibitory effect on the reproduction of Daphnia magna at a TWM concentration 3.7 mg solids/L after 21 days of exposure (NOEC). The 21 -d NOEC for growth reduction was 1.6 mg solids/L (TWM concentration).
Executive summary:

The long-term toxicity of the test item to aquatic invertebrates was studied in a Daphnia magna reproduction study according to OECD 211 and under GLP conditions.

10 neonates (<24 h old) were individually exposed in a semi-static sytem to nominal test concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg solids/L for 21 days with test solutions renewed every 48 hours. Additionally, a blank control was included with 20 neonates. Parental mortality, number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded and the lengths of the surviving parental daphnids were measured at the end of the test.

Samples taken at the beginning and the end of three 48-hour renewal intervals were analyzed. The concentrations measured in the freshly prepared solutions ranged between 47 - 133% of nominal, with the majority of results being within 87 -120% of nominal. Time Weighted Mean concentrations were calculated since the concentrations appeared to be unstable during the refreshment period, and were 0.075, 0.15, 0.45, 1.6 and 3.7 mg solids/L.

Mortality in the controls did not exceed 20%, mortality in the test item groups ranged from 10 to 40% but was not statistically different from the control treatment. An increase of reproduction, rather than a reduction was observed in all groups tested. The onset of reproduction was not delayed in any of the test concentrations when compared to the controls (onset at day 7), except at the highest treatment group (at day 8). The change in mean body length of parent daphnids ranged between -1.0 and 1.8% in the four lowest concentrations and was not dose-related. At the highest concentration, a statistically significant reduction of 4.6% was observed.

The 21 -d NOEC for reproduction of Daphnia magna exposed to the test item was 3.7 mg solids/L (TWM concentration). The 21 -d NOEC for growth reduction was 1.6 mg solids/L (TWM concentration).

The study met all validity criteria and is considered reliable without restrictions.

Description of key information

The test was performed with a substance analogue. The rationale to read across the data is attached in section 13.

The long-term toxicity of the test item to aquatic invertebrates was studied in a Daphnia magna reproduction study according to OECD 211 and under GLP conditions.

10 neonates (<24 h old) were individually exposed in a semi-static sytem to nominal test concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg solids/L for 21 days with test solutions renewed every 48 hours. Additionally, a blank control was included with 20 neonates. Parental mortality, number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded and the lengths of the surviving parental daphnids were measured at the end of the test.

Samples taken at the beginning and the end of three 48-hour renewal intervals were analyzed. The concentrations measured in the freshly prepared solutions ranged between 47 - 133% of nominal, with the majority of the results being between 87 - 120%. Time Weighted Mean concentrations were calculated since the concentrations appeared to be unstable during the refreshment period, and were 0.075, 0.15, 0.45, 1.6 and 3.7 mg solids/L.

Mortality in the controls did not exceed 20%, mortality in the test item groups ranged from 10 to 40% but was not statistically different from the control treatment. An increase of reproduction, rather than a reduction was observed in all groups tested. The onset of reproduction was not delayed in any of the test concentrations when compared to the controls (onset at day 7), except at the highest treatment group (at day 8). The change in mean body length of parent daphnids ranged between -1.0 and 1.8% in the four lowest concentrations and was not dose-related. At the highest concentration, a statistically significant reduction of 4.6% was observed.

The 21 -d NOEC for reproduction of Daphnia magna exposed to the test item was 3.7 mg solids/L (TWM concentration). The 21 -d NOEC for growth reduction was 1.6 mg solids/L (TWM concentration). The study met all validity criteria and is considered reliable without restrictions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.6 mg/L

Additional information

Key value is based on the lowest NOEC found in the study (growth reduction).

21 -d NOEC for growth reduction: 1.6 mg/L, based on analytically confirmed test substance corrected for water content (and specified in the report as "solids")

21 -d NOEC for reproduction: 3.7 mg/L, based on analytically confirmed test substance corrected for water content (and specified in the report as "solids")