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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:
2017-03-22 to 2017-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
No major (The temperature was not constant within ± 1°C. The pH varied by more than 1.5 units. The test is still valid, as it was shown that validity criteria are met).
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10.0, 3.13, 0.977, 0.305, 0.0954 mg/L (nominal)
- Sampling method: For verification of the test item concentrations and stability of the test item, samples from the freshly prepared test solutions and aged test solutions were taken from all concentration levels and the controls at test start, at one test solution renewal in the second week and at one test solution renewal during the third week. Analytical samples were taken from all test item concentrations and control at test start, after 9 days and 16 days from fresh solutions and after 2 days, 12 days and 19 days from aged solutions.
- Sample storage conditions before analysis: 900 µL samples were taken and stabilized with 300 µL acetonitrile (F1-F3; F = frozen) and 7.50 mL samples were taken and stabilized with 2.5 mL acetonitrile (F4-F5). All samples were stored deep frozen.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 10 mg of the test item for preparing the stock solution S1 was weighed on a weighing scoop and transferred to a volumetric flask (1000 mL). M4 medium was added up to the bench mark and the solution was treated with 15 minutes of ultrasonication. Lower test solutions were prepared by dilution of the appropriate stock solutions with test medium. The preparation procedure was repeated every Monday, Wednesday and Friday. About 50 mL of the prepared solutions were transferred to each test vessel.
- Controls: M4 medium without test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, the solution was clear and transparent.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Great water flea
- Strain/clone: Clone V
- Source: Federal Environment Agency in Berlin/Germany
- Age of parental stock (mean and range, SD): Animals are continuously bred in the laboratory. Freshly hatched daphnids less than 24 hours old were used for the test.
- Feeding during test: yes
- Food type: Unicellular green algae (Desmodesmus subspicatus, formerly Scenedesmus subspicatus)
- Amount: 0.1 mg carbon / daphnia / day from day 1 to 7, 0.15 mg carbon / daphnia / day from day 8 to 14 and 0.2 mg carbon / daphnia / day from day 15 to 21. Additional nutrients (100 µL of supernatant of cooked Tetra Min) were provided every Monday, Wednesday and Friday
- Frequency: Algae were given daily, Nutrients were given every Monday, Wednesday and Friday

ACCLIMATION
- Acclimation conditions (same as test or not): Same as test
- Health during acclimation (any mortality observed): No mortality observed

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
A pipette was used to separate the young daphnids from the adults.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
12 - 14°dH
Test temperature:
19.8 - 21.7°C (Overall mean 20.1°C ± 0.7°C Std. Dev.)
pH:
7.41 - 9.26 (Overall mean 8.37 ± 0.66 Std. Dev.)
Dissolved oxygen:
8.9 - 12.7 mg/L (Overall mean 10.2 mg/L ± 1.5 mg/L Std. Dev.)
Nominal and measured concentrations:
Nominal: 10.0, 3.13, 0.977, 0.305, 0.0954 mg/L and control
Measured: 2.85, 0.60, 0.27, 0.12, 0.05 mg/L and control
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL filled with 50 mL test solution. The test units were covered with a glass plate
- Aeration: None
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10, 1 additional replicate for each test item concentration without food and daphnids
- No. of vessels per control (replicates): 10, 1 additional replicate for control without food and daphnids

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 medium according to guideline
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours photoperiod /8 hours darkness daily
- Light intensity: 1200 - 1350 lux

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.19
- Range finding study: No
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 2.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.115 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.268 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 2.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Alive offspring produced by adults alive at test end.
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Alive offspring produced by adults alive at test end.
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 2.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Alive offspring produced by adults alive at test end.
Details on results:
- Behavioural abnormalities: Not given.
- Mortality of control: 0% mortality
- Biological Results: The first offspring in the control was observed on day 9. At test item concentrations of 0.0954, 0.305 mg/L and 10.0 mg/L first offspring was observed on day 8. At 0.977 mg/L first offspring was observed on day 9.
The first offspring at the concentration level 3.13 mg/L was observed on day 11.
Reported statistics and error estimates:
For the calculation of NOEC, LOEC and EC50 SAS® Proprietary Software Version 9.3. was used. The significance level for all statistical tests was p < 0.05.
Mortality of adult Daphnia:
Due to a lack of mortality no statistical evaluation was performed with this parameter.

Reproductive output per parent animal from test start:
A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic, a test for homogeneity of the data was performed according to Levene. The NOEC and LOEC were determined by using a multiple comparison method (Jonckheere-Terpstra). The EC50 of reproduction (alive offspring per alive adult at test start) was determined using probit analysis following Gompertz procedure for nominal test item concentration and by using probit analysis following normal distribution for actual concentrations.

Reproductive output per parent animal alive at test end:
A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic, a test for homogeneity of the data was performed according to Levene. The NOEC and LOEC were determined by using a multiple comparison method (Dunnett`s t-test, left sided). The inhibition of reproduction (alive offspring per alive adult at test end) was below 50 % and therefore no statistical evaluation was performed to determine the EC50 of this parameter.

Table 1: Mortality of adult animals for every day (% of initial numbers)

Conc./Day

1

2

3

4

5

6

7

8

9

10

 

[mg/L]

 

 

 

 

 

 

 

 

 

 

Control

0

0

0

0

0

0

0

0

0

0

0.0954

0

0

0

0

0

10

10

10

10

10

0.305

0

0

0

0

0

0

0

0

0

0

0.977

0

0

0

0

0

0

0

0

10

10

3.13

0

0

0

10

10

10

10

10

10

10

10.0

0

0

0

0

0

0

0

0

0

0

 

Conc./Day

11

12

13

14

15

16

17

18

19

20

21

[mg/L]

 

 

 

 

 

 

 

 

 

 

 

 

Control

0

0

0

0

0

0

0

0

0

0

0

0.0954

10

10

10

10

10

10

10

10

10

10

10

0.305

10

10

10

10

10

10

10

10

20

20

20

0.977

20

30

30

30

30

30

30

30

30

30

30

3.13

10

20

30

30

30

30

30

30

30

30

30

10.0

10

20

30

30

30

30

30

30

30

30

30

 

Table 2: Sum of alive offspring per alive adult at the end of the test as inhibition to control

Conc. [mg/L]

Mean number of alive

offspring per alive adult

at test end

Inhibition

Control

[%]

 

 

 

Control

78.5

-

0.0954

78.4

0.1

0.305

88.6

-12.9

0.977

85.0

-8.3

3.13

54.4

30.7

10.0

62.7

20.1

- not calculated

Table 19: Determined concentration of ND

Test item nominal [mg/L]

Time weighted

average

[% of nominal]

Test item actual [mg/L]

Control

-

-

0.0954

48.7

0.0464

0.305

37.7

0.115

0.977

27.5

0.268

3.13

19.2

0.600

10.0

28.5

2.85

- not calculated

Validity criteria fulfilled:
yes
Conclusions:
The lowest observed effect value for mortality of adults and reproduction was a NOEC of 0.305 mg/L (nominal) equivalent with 0.115 mg/L (measured).
Executive summary:

The toxicity of Nonane-1,9 -diol on the reproduction of Daphnia magna was determined in a reliable GLP study according to OECD 211. Individuals of D. magna were exposed to nominal test concentrations of 10.0, 3.13, 0.977, 0.305, 0.0954 mg/L (plus control), which are equivalent to 2.85, 0.60, 0.27, 0.12, 0.05 mg/L (control not calculated) measured concentrations for 21 days. Concentrations and stability of the test substance were analytically verified via LC-MS. Observations were made on mortality and reproduction of D. magna. The lowest observed effect value for mortality of adults and reproduction was a NOEC of 0.305 mg/L (nominal), equivalent with 0.115 mg/L (measured).

Description of key information

Mortality

NOEC (21 d, D. magna): 0.115 mg/L (measured)

LOEC (21 d, D. magna): 0.268 mg/L (measured)

LC50 (21 d, D. magna): > 2.85 mg/L (measured)

Reproduction (at test end)

NOEC (21 d, D. magna): 2.85 mg/L (measured)

LOEC (21 d, D. magna): > 2.85 mg/L (measured)

LC50 (21 d, D. magna): > 2.85 mg/L (measured)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.115 mg/L

Additional information

The toxicity of Nonane-1,9 -diol on the reproduction of Daphnia magna was determined in a reliable GLP study according to OECD 211. Individuals of D. magna were exposed to nominal test concentrations of 10.0, 3.13, 0.977, 0.305, 0.0954 mg/L (plus control), which are equivalent to 2.85, 0.60, 0.27, 0.12, 0.05 mg/L (control not calculated) measured concentrations, for 21 days. Concentrations and stability of the test substance were analytically verified via LC-MS. Observations were made on mortality and reproduction of D. magna. The lowest observed effect value for mortality of adults and reproduction was a NOEC of 0.305 mg/L (nominal), equivalent with 0.115 mg/L (measured).

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