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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Cited as OECD Guide-line 202, part 2 (Daphnia sp., Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution 7.6 mg Diuron/L was prepared by ultrasonification and stirring
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna Straus
- Source: Strain from Bundesgesundheitamt in Berlin, Germany, Genotype no 2/Type B
- Age of parental stock (mean and range, SD): the first instars used in the test were obtained by screening of adults; only parents of the same age (14-21 days) ±12 hours were used
- Age of test animals: less than 24 h old
- Feeding during test: yes
- Food type: Scenedesmus subspicatus (green algae).
- Amount: 1x 10 8 cells/L, on Fridays 3x 10 8 cells/L
- Frequency: daily

ACCLIMATION
- Acclimation period: The strain has been maintained in the testing laboratory for more than 10 years
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Scenedesmus subspicatus (green algae), occasionally commercial ornamental fish food (Trade name: TetraMin®).
- Feeding frequency: daily
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
not given
Test temperature:
19.8 – 20.1°C
pH:
Fresh solutions: 8.0 – 8.2
Aged solutions: 7.8 – 8.1
Dissolved oxygen:
Fresh solutions: 8.3 – 9.6 mg/L
Aged solutions: 8.1 – 9.7 mg/L
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 0,0.032, 0.056, 0.10, 0.18, 0.32, 0.56, 1.0 nad 1.8 mg/L
Mean measured concentrations ranged from 93 to 102 % of the nominal
Measured concentrations: 0, 0.033, 0.054, 0.096, 0.17, 0.32, 0.56, 0.97 and 1.75 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass beakers
- Material, size, headspace, fill volume: 200ml of test solution
- Aeration: none
- 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
Note: 3 further vessels containing 5 daphnids each were included to monitor survival/mortality

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7-medium” similar to “M4-medium (see Table 1)
- Conductivity: 580 µS/cm
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8, light/dark caycle
- Light intensity: 700 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobility (e.g. mortality) of adult daphnids
- Number of living young daphnids
- Number of dead young daphnids
- Body weight and length of parent after 21 days.
The number of offspring was determined daily from day 7 – 12. Thereafter, tree times per week on the occasion of medium exchange

RANGE-FINDING STUDY
- Test concentrations: 1.0, 1.8, 3.2, 5.6 and 10 mg/L (30 animals per concentration)
- Results used to determine the conditions for the definitive study: Yes; results indicated an EC50 of 1.4 mg/L.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.56 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.97 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: effect on body weight of parent animal
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: effect on body weight of parent animal
Details on results:
- Mortality of parent animals: No treatment related mortality was observed (Mortality during test duration is given in Table 2) No EC50 could be determined
- No. of offspring produced per day per female: Effects on reproduction was observed at concentrations of 0.97 mg Diuron/ and higher (see Table 3)
- Body length and weight of parent animals: Reduced parental body dry weight and length.
Reported statistics and error estimates:
For testing of normality the Kolmogoroff-Smirnoff-Test and for homogeneity of variance the Bartlett’s test was used. For normal distributed data and ANOVA followed by a Dunnett’s test. For the number of offspring per parent the data were tested using Mann and Withney U-test, since the variance was not homgeneous.

Table 2.              Effect data (survival)

Concentration
(mg Diuron/L)
(
nominal)

Cumulative mortality (%)

Time (days)

0

5

7

10

14

16

21

Control

0

0

0

0

0

7

7

0.033

0

0

0

0

7

7

7

0.054

0

0

0

0

0

0

0

0.096

0

0

0

0

0

0

0

0.17

0

0

0

0

0

0

0

0.32

0

7

7

7

7

7

7

0.56

0

0

0

0

0

0

0

0.97

0

7

7

7

7

7

7

1.75

0

0

0

0

0

7

7

Table 3.            Effect data (Reproduction)

Concentration
(mg Diuron/L)
(
nominal)

Reproduction of parent daphnids

Time (days)

Cumulated offspring on day 21

Day of first reproduction
(mean)

Number of reproduction days

Mean number of offspring per reproduction day

Mean body length (mm)

Mean body weight (mg)

Control

107.8

7.9

14.1

7.60

4.59

1.065

0.033

97.7

8.6

13.4

7.29

4.63

1.094

0.054

101.1

8.0

14.0

7.22

4.61

1.004

0.096

91.3

9.0

13.0

7.03

4.61

0.976

0.17

90.0

8.3

13.7

6.55*

4.57

0.833*

0.32

92.9

8.3

13.7

6.75

4.46

0.791*

0.56

96.9

7.9

14.1

6.88

4.41*

0.637*

0.97

81.4*

8.5

13.5

6.02*

4.22*

0.635*

1.75

35.4*

9.7

12.3

2.92*

3.74*

0.476*

* significantly different from control

Table 4.    Validity criteria for invertebrate reproduction test according to OECD Guideline 202

 

Fulfilled

Not fulfilled

Mortality of Daphnia in the control < 10%

x

 

Dissolved oxygen concentration > 60 % at the end of the test

x

 

Mortality of parent animals < 20% at test termination

x

 

Concentration of test substance³80% of initial concentration during test

x

 

First young born in controls after a max. of 9 days

x

 

Mean cumulative number of young per female in the controls = 20 at a temperature of 20 ± 1°C

x

 

Mean number of live offspring produced per parent animal surviving at test termination³60

x

 

 

Executive summary:

A long term toxicity test with Daphnia magna, according to OECD guideline 211 (cited as OECD guideline 202, part 2) with GLP, is available for this test substance. This study is based on measured concentrations of the test substance and calculated the NOEC (21d) to be 0.56 mg/L, with a LOEC of 0.97 mg/L. Further to this the NOEC and LOEC values for effects on the parent animals were calculated at 0.1 and 0.17 mg/L respectively. Therefore the test substance is found to be a chronic toxicity hazard to aquatic invertebrates, evidenced by the NOEC/LOEC values of < 1 mg/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Cited as OECD Guide-line 202, part 2 (Daphnia sp., Reproduction Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on test solutions:
10 mg of the test substance were added to 1 L test medium, final stock solution (10 µg/ml) was stored in the refrigerator until use. On treatment days, dilutions of the stock were performed to obtain the nominal concentrations. Test concentrations were based on the EC50 (48 h) of 1.4 mg/L
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Waterflea
- Source: laboratory culture
- Feeding during test:
on days 0, 2, 4, 7, 9, 11, 14, 16 and 18
fed with a mix of yeast and algae (Scendesmus subspicatus)
Days 0, 2 and 4 fed 200,000 cells/ml yeast and 200,000 cells/ml algae;
days 7, 9 and 11 fed 300,000 cells/ml yeast and 300,000 cells/ml algae;
days 14, 16 and 18 fed 600,000 cells/ml yeast and 600,000 cells/ml algae

ACCLIMATION
- Acclimation period: Bred at lab under standard conditions
- Acclimation conditions: same as test conditions


Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
21 d
Test temperature:
21.5-22.5 °C
pH:
8.0-8.2
Dissolved oxygen:
8.9 – 9.3 mg oxygen/L
Nominal and measured concentrations:
Nominal: 0, 0.0003, 0.0016, 0.008, 0.04, 0.2 and 1.0 mg/L
Test concentrations were based on the EC50 (48 h) of 1.4 mg/L
Analysis of the test article showed that for a target concentration of 0.04 mg/L the mean concentrations ranged from 89.7% - 91.8% of the nominal value. For the target concentration of 1.0 mg/L the range was 88.0% - 105.0% of the nominal value
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Aeration: aerated until oxygen saturation prior to test
- Renewal rate of test solution (frequency/flow rate): on days 0, 2, 4, 7, 9, 11, 14, 16 and 18
- No. of organisms per vessel: Initially 10 daphnids per beaker, After 9 days one daphnid selected and placed in one 50 ml beaker
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: standard reconstituted water (see table 1.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12-16 h/day
- Light intensity: ca. 500-2000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and reproduction

RANGE-FINDING STUDY
- Test concentrations: Test concentrations were based on the EC50 (48 h) of 1.4 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality of parent animals: The mortality of the control samples was 10%, primarily due to mechanical damage during renewal procedures and the test is considered to be valid based on these findings. From day 0 to day 7 no mortality was observed at the lowest concentrations. At concentrations of 0.008 up to 1.0 mg/L a mortality rate of 10 -25% was observed.
- No. of offspring produced per day per female: Reproduction started from day 11 of the exposure period, no significant influence of the test substance was observed up to the highest concentrations. The mean number of daphnids alive per adult in the untreated control was 48

Table 2. Influence of Technical Diuron on the total (cumulative) number of alive, young daphnids reproduced by surviving adults

Exposure Day

Concentration of test substance (nominal)

mg/L

 

0

0.0003

0.0016

0.008

0.04

0.2

1.0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

4

0

0

0

0

0

0

0

7

0

0

0

0

0

0

0

9

0

0

0

0

0

0

0

11

66

67

37

43

12

40

24

14

131

159

167

147

143

127

79

16

193

202

370

275

322

311

99

18

311

276

450

385

372

425

130

19

338

295

493

489

427

435

188

20

409

317

493

489

421

436

207

21

476

421

524

522

426

460

324

%-control

100

88.4

110.0

109.7

89.5

96.6

68.1

Executive summary:

A long term toxicity test with Daphnia magna, according to the prevailing OECD guidelines (OECD 211, cited as OECD guideline 202, part 2) with GLP, is available for this substance. Since no significant effects could be observed up to the highest concentration of 1 mg/L, the NOEC was determined as=1 mg/L and the LOEC as > 1 mg/L.

Description of key information

Diuron is a chronic (long-term) effect to aquatic invertebrates

Key value for chemical safety assessment

Additional information

Two long term toxicity tests with Daphnia magna, according to OECD guideline 211 (cited as OECD guideline 202, part 2) with GLP, are available for Diuron. The key study is based on measured concentrations of the test substance and calculated the NOEC (21d) to be 0.56 mg/L, with a LOEC of 0.97 mg/L. Further to this the NOEC and LOEC values for effects on the parent animals were calculated at 0.1 and 0.17 mg/L respectively (Heimbach, 1996). The supporting study could determine no significant effects up to the highest concentration of Diuron tested, therefore the NOEC was determined as =1 mg/L and the LOEC as > 1 mg/L in this case (Ritter, 1989). Based on these findings, the results from the key study (Heimbach, 1996) should be used to determine that the test substance is a chronic toxicity hazard to aquatic invertebrates, evidenced by the NOEC/LOEC values of < 1 mg/L.

Discussion on endocrine disruption:

As at the current stage there is still no agreed approach and guidance on how to evaluate endocrine disruption in various EU legislations the OECD Conceptual framework for Testing and Assessment of Endocrine Disruptors might be used. Comparing available data with the Framework`s level concept allowed to rank Heimbach et al. (1996) and Ritter (1989) in OECD level 5, which means both studies allow final conclusion on endocrine disruption properties of diuron. Both studies investigated on effects to reproduction of Daphnia magna according to GLP standard and formerly OECD test guideline No. 202, part II, which mainly translated into the current OECD test guideline 211 on reproduction of D. magna. Both studies reported of no effect to daphnia reproduction which is the endpoint of OECD test guideline 211. There is, thus, no indication that diuron could be an endocrine disruptor. See also the document on the ad hoc assessment on endicrine disruption properties of diuron attached in chapter 13 of IUCLID.