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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
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)
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Qualifier:
equivalent or similar to guideline
Guideline:
other: ISO 10706
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations
- Sampling method: The concentration of the test substance was determined in one representative test interval per week in each freshly prepared test solution (test vessels without daphnids) and in the 48 hour or 72 hour old test solutions before renewal (combined sample from test vessels with
daphnids).
- Sample storage conditions before analysis: the samples were analyzed immediately
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The necessary volume of the test substance was calculated from the density. Prior to use the test substance container was shaken to ensure homogeneity. The stock solution (20 mg/L) was prepared by pipetting 40 mg (42.1 µL based on the density) or 100 mg (105.3 µL) test substance into 2 L or 5 L of test medium and stirring for approximately 10 min. The stock solution was checked for the presence of any undissolved substance before diluting this stock solution to reach the lower test concentrations. By dilution of this stock solution with M4 medium the test concentrations were prepared.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No. The stock solution appeared colorless-clear.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: Institut National de Recherche Chimique Appliquée (France, 1978), cultured continuously in test facility since 1978
- Age of parental stock (mean and range, SD): maximum 4 weeks
- Feeding during test: yes
- Food type: live green algae Desmodesmus subspicatus
- Amount: Day of test: 0-3 (0.10 mg TOC), 4-5 (0.12 mg TOC), 6-7 (0.14 mg TOC), 8-21 (0.20 mg TOC)
- Frequency: daily

ACCLIMATION
- Acclimation conditions (same as test or not): same as test
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
2.20 - 3.20 mmol/L
Test temperature:
20°C (±1°C)
pH:
7.5 - 8.5
Dissolved oxygen:
≥ 3mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 2.7, 3.7, 5.2, 7.2, 10 mg/L
Measured (mean measured concentrations): 0 (control), 2.20 (81.5%), 3.09 (83.5%), 4.77 (91.7%), 6.79 (94.3%), 9.82 mg/L (98.2%)
(see detailed results in "any other information on results incl. tables")
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers (100 mL nominal volume)
- Type: open (covered with petri plates)
- Aeration: none
- Material, size, headspace, fill volume: 50ml headspace, fill volume 50 mL
- Renewal rate of test solution (frequency/flow rate): semi static / 3 times per week
- No. of organisms per vessel: 1 parent
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 1 animal / 50 mL

OTHER TEST CONDITIONS
- Photoperiod: 16 light : 8 dark
- Light intensity: 620 -670 lux at a wave length of 400 - 750 nm
- Adjustement of pH: all stock solutions were adjusted with 1 molar HCl to a pH of approx. 8

TEST MEDIUM / WATER PARAMETERS
- pH: 7.5 - 8.5
- Total organic carbon: < 2mg/L
- Ca/mg ratio: about 4:1
- Conductivity: 550 - 650 µS/cm

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: In a preliminary Daphnia reproduction test a NOEC = 4.13 mg/L and LOEC = 9.09 mg/L was found (experimental conduct in accordance with GLP but without a GLP status). These results guided the concentration selection for the definitive test.
Reference substance (positive control):
yes
Remarks:
sodium chloride
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
(analytically verified)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
7.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
(analytically verified)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
(analytically verified)
Basis for effect:
mortality
Remarks:
(parental mortality)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
7.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
(analytically verified)
Basis for effect:
mortality
Remarks:
(parental mortality)
Details on results:
- Mortality of parent animals: control: 0%
- Mean living young per surviving adult after 21 d: control: 167
- Time to first brood release or time to hatch: control: 8.2 d
- Effect concentrations exceeding solubility of substance in test medium: no
- The analytically measured concentrations of the test substance in the test water were within the range of ± 20% of the nominal concentration in all tested concentrations with the only exception that in aged test solutions concentrations decreased slightly with the largest decrease
in the lowest treatment group, which was 79% of the mean initial value. However, mean measured concentrations for the whole exposure period were within ±20% of the nominal concentration. Since both mean initial and overall mean concentrations were acceptable, the nominal concentrations can be used to evaluate the results of the study.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (48h): 5.08 g/L (test date: 05 Mar 2013)
- Other: - remarks: normal response to the reference substance (NaCl)

EFFECT VALUES

Effect concentration [mg/L]

Endpoint

nominal

measured

(arithm. mean)

NOEC

LOEC

NOEC

LOEC

Reproduction

7.20

10.00

6.79

9.82

Mortality

7.20

10.00

6.79

9.82

 The data were not sufficient to calculate any ECx values.

ANALYTICAL CONCENTRATION CONTROL

Nominal concentration

[mg/L]

Analytically measured concentration

Mean initiald

Mean 48/72h old

arithm. mean

[mg/L]

[%]a

[mg/L]

[%]b

[mg/L]c

[%]a

0 (control)

<LOQe

<LOQ

<LOQ

2.7

2.46

91

1.95

79

2.20

82

3.7

3.33

90

2.86

86

3.09

84

5.2

4.93

95

4.60

93

4.77

92

7.2

6.93

96

6.65

96

6.79

94

10

9.93

99

9.70

98

9.82

98

a: % of nominal concentration

b: % of mean initial

c: based on mean-initial value and mean-old value

d: initial measured is the mean measured concentration at the start of each renewal period

e: Limit of quantification (0.3 mg/L)

BIOLOGICAL RESULTS

Reproduction and Mortality Summary after 21 days

Nominal concentration [mg/L]

Reproduktion

Mortality

per surviving parent

per introduced parent

Parent animals

Mean living young

% effectb

Mean living young

% effect

0.0

167 (5.9%)a

-

167 (5.9%)

-

0

2.7

184

-

184

-

0

3.7

192

-

192

-

0

5.2

169

-

169

-

1

7.2

168

-

168

-

0

10.0

149*

11

124**

26

2*

* : p≤ 0.05; ** : p≤ 0.01

a: Coefficient of variation for control fecundity based on surviving parent.

b: Effect relative to control. (Only calculated for statistically significant effects.)

Other biological observations among surviving parent animals in each concentration after a 21 day exposure

Test conc.

[mg/L, nominal]

Mean Growth

Length[mm]

Immobile young

[%]

Mean days

to first brood

Aborted eggs

[%]

0 (control)

4.51

0.2

8.2

1.0

2.7

4.59

0.3

8.0

0.7

3.7

4.30*

0.1

8.2

0.6

5.2

4.31

0.2

8.4

1.0

7.2

4.20**

0.7

8.1

0.9

10

3.23**

0.8

8.4

1.8

*: p ≤ 0.05; **: p ≤ 0.01 

No additional adverse effects or abnormal behavior were observed in any of the test groups.

OTHER OBSERVATIONS pH-values (always measured in replicate 1)

Nominal concentration [mg/L]

Day of the test

0a

3b

10a

12b

17a

19b

0 (control)

8.0

8.1

7.9

8.0

7.9

7.9

2.7

8.0

8.3

8.0

8.0

8.0

7.9

3.7

8.1

8.2

7.9

8.1

8.0

8.0

5.2

8.0

8.3

8.1

8.1

8.0

8.0

7.2

8.1

8.3

8.1

8.2

8.0

8.1

10

8.1

8.2

8.0

8.2

8.1

8.2

a: in the freshly prepared test solution

b: before renewal in the old test solutions

Oxygen content [mg/L] (always measured in replicate 1)

Nominal concentration [mg/L]

Day of the test

0a

3b

10a

12b

17a

19b

0 (control)

9.0

9.1

8.9

9.2

8.7

8.4

2.7

9.1

9.3

9.0

9.1

8.8

8.4

3.7

9.1

9.3

8.9

9.2

8.8

9.0

5.2

9.0

9.2

9.0

9.2

8.8

8.9

7.2

9.1

9.3

9.0

9.1

8.8

8.9

10

9.1

9.3

9.0

9.3

8.7

9.0

a: in the freshly prepared test solution

b: before renewal in the old test solutions

Total hardness [mmol/L] (always measured in replicate 1)

Nominal concentration [mg/L]

Day of the test

0a

3b

10a

12b

17a

19b

0 (control)

2.41

2.45

2.43

2.46

2.45

2.46

2.7

2.43

2.45

2.43

2.46

2.45

2.48

3.7

2.41

2.43

2.41

2.45

2.45

2.46

5.2

2.43

2.45

2.41

2.46

2.45

2.48

7.2

2.41

2.43

2.43

2.46

2.45

2.48

10

2.41

2.43

2.41

2.46

2.45

2.50

a: in the freshly prepared test solution

b: before renewal in the old test solutions

Instantaneous temperature [°C]

Temperature was measured continuously during the exposure period in a

separate water filled vessel proximal to the test vessels. The temperature ranged from 19.7-20.4°C.

Validity criteria fulfilled:
yes

Description of key information

Chronic effects are not expected.

Key value for chemical safety assessment

Additional information

To determine chronic effects of N4-Amine N,N’-Bis-(3-Aminopropyl)-ethylenediamine (CAS 10563 -26 -5) on aquatic invertebrates, a GLP-study according to OECD 211 was performed with Daphnia magna (21 d-reproduction test). The test concentrations were analytically verified. The mean measured concentrations for the whole exposure period were within ±20% of the nominal concentration. The NOEC was determined to be 7.2 mg/L (reproduction; nominal) (BASF SE, 2014; report no. 51E0402/04E011). All validity criteria were fulfilled in this study. Based on the NOEC of 7.2 mg/L chronic effects on aquatic invertebrates are not expected.