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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:
2008-07-30 - 2008-08-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sample storage conditions before analysis: Room temperature, protected from moisture and light
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
- Age at study initiation: 2-24 h old
- Method of breeding:
Culture: In 2 - 3 L glass vessels with approximately 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength
max. 20 µE.m-2 . s-1.
-Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
- Feeding during test:
- Food type: mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris
- Amount: , with an algae cell density of > 106 cells/mL.
- Frequency: At least 5 times per week

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
160 to 180 mg CaCO3/L
Test temperature:
The temperature in the test vessels was within the range 21.1 to 22.0 °C

pH:
The measured initial and final pH values of the control and the highest tested concentration in one replicate per test and control group are shown in Table 17. Due to 100% mortality at the test concentration 400 mg/L on day 4, the last values were taken from one replicate of the test concentration 200 mg/L.

Deviations of < 1.5 units between initial and final pH values are regarded as being not significant. The initial pH-values varied from 9.36 to 9.61 at the test concentrations 200 and 400 mg/L and from 7.61 to 7.88 at the control. The final pH-value varied from 8.41 to 8.46 at the test concentrations 200 and 400 mg/L and from 7.41 to 7.61 in the control.

Dissolved oxygen:
The final oxygen concentrations of the measured replicates in the test media of the concentrations 200 and 400 mg/L and the control were found to be >= 7.15 mg/L

Nominal and measured concentrations:
Nominal concentrations of Dimethylaminopropyl methacrylamide were selected based on the results of an acute immobilization test (Lab ID 080509RA/DAI12401) as follows: 25.0 - 50.0 - 100 - 200 - 400 mg/L. All concentrations of test item Dimethylaminopropyl methacrylamide and control were analytically verified at three sampling dates via LC-MS/MS of samples on days 0, 7, 19 (fresh media, 0 h) and on days 2, 9, 21 (old media, 48 h). The recoveries were in the range of ± 20 % of the nominal values in the fresh and old media. Analytical results are given in Table 24 and Table 25. The results of the chemical analyses show that the test organisms were fully exposed to the test item during the test. Therefore, all effect values are given based on the nominal concentrations.
Details on test conditions:
Stock solution: The stock solution (400 mg/L test item was weighed out) was freshly prepared with dilution water.
Dispersion treatment: Agitation
Nominal test item concentrations: 25.0 - 50.0 - 100 - 200- 400 mg/L (factor 2)

CONTROL: 10 replicates of dilution water without test item
TEST METHOD: Semi-static, medium renewal at least 3 times per week
Test duration: 21 d
Test vessels: Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity
Volume of the test medium: 50 mL
Dilution water Same as for culture
Loading: 10 animals, individually held
Number of replicates: 10 per concentration and control
Age of test organisms 2 to 24 h old daphnids were used for the definitive study. They at test begin were obtained by removing the mother animals twice within 22 h.
Feeding: At least 5 times per week. Per test vessel Pseudokirchneriella subcapitata (0.670 - 1.050 mL) and Desmodesmus subspicatus (1.060 - 1.795 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day. Variation was according to the density of the algae suspension, but was the same for all test groups on each feeding day.
Test temperature: 18 - 22 °C, ± 2 °C constant
Illumination Max. 20 µE.m-2 .s-1
Photoperiod 16/8 h light/dark cycle

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
94.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 77.1 - 117 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
141 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: adult mortality
Remarks on result:
other: 100 - 200 mg/L
Details on results:
- The average number of juveniles per parent in the control group was 121 after 21 days. At the concentration levels 200 and 400 mg/L no juveniles occurred due to the mortality of all parent animals. The reproductive output was statistically significant reduced at the concentration levels 50.0 and 100 mg/L. The concentration level 25.0 mg/L was not statistically significant reduced when compared to the control (One Way Analysis of Variance, DUNNETT’S method, p = 0.05). For details see Table below

- The coefficient of variation of the number of living offspring produced per parent in the control group was 6 % .

- The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance, DUNNETT’S method (p = 0.05). There was a statistical significance at the concentration level 100 mg/L when compared to the control. For concentration levels 200 and 400 mg/L no intrinsic rates were calculated due to the mortality of all parent animals. For details see Table below.

- No stillborn juveniles and aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles came to a maximum of 2 % at the concentration level 100 mg/L .

- The first brood occurred on day 8 at all parent animals of the control group and the concentration levels of 25.0 and 50.0 mg/L. At the tested concentration level 100 mg/L the mean day of release of the first brood was 9.4. At seven of ten parent animals the first brood was released on day 10. Release of the first brood after day 9 is regarded to be too late and biologically significant, if it occurs at several daphnids of the test group (Table 8).
Five broods were released by all surviving animals of control group and the concentration levels of 25.0 and 50.0 mg/L. At concentration level 100 mg/L the number of broods was reduced in comparison to the control group at seven of ten parent animials. There were three animals releasing five broods, six animals releasing four broods and one animal releasing three broods.

A summary of test item related effects concerning the adult mortality and growth (weight, length) is given in the table below.

- At the tested concentration levels 200 and 400 mg/L the adult mortality amounted to 100 % after 21 days. The test item did not induce significant mortality (<=20 %) of parent animals at any of the other concentrations and at the control. At the control group as well as at the test concentrations 25.0 to 100 mg/L all daphnids survived until the end of the study. The EC50 for adult mortality after 21 days was calculated to be 141 mg/L.

- The dry body weight of the parental daphnids in the tested concentration levels 25.0 and 50.0 mg/L were comparable to the control group. The mean dry body weight in these concentration levels ranged from 0.85 to 0.97 mg per daphnid. The mean dry body weight in the control was 0.89 mg per daphnid. At the concentration level 100 mg/L the mean dry body weight of 0.57 mg per daphnid (reduction of 36 %) was significantly reduced in comparison to the control group.
The mean value of the total body length in the tested concentration level 25.0 was 5.25 mm per daphnid and comparable to the control group with 5.30 mm per daphnid. At the concentration levels 50.0 and 100 mg/L the total body length of 4.88 and 4.20 mm per daphnid was significantly reduced in comparison to the control group (One Way Analysis of Variance, DUNNETT’S method, p = 0.05).

- No males and ephippia (winter eggs) occurred in control or test groups.



Results with reference substance (positive control):
The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence interval (CI) after 24 h of the reference item in mg/L was determined by sigmoidal dose-response regression:
EC50 : 1.75 (CI 1.65 - 1.85)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.
Reported statistics and error estimates:
see: Remarks on results and error estimates

Test Item Related Effects on Reproduction, NOEC, LOEC and EC50

Effects

Nominal Concentration [mg/L]

Control

25.0

50.0

100

200

400

Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)

121 ± 7

126 ± 12

105 ± 171)

56 ± 161)

---

---

Mean Intrinsic Rates of Natural Increase

0.50

0.51

0.49

0.421)

---

---

Appearance of First Brood [Mean Day]

8.0

8.0

8.0

9.42)

---

---

Mean Number of Broods

5.0

5.0

5.0

4.23)

---

---

NOECReproduction

25.0 mg/L

LOECReproduction

50.0 mg/L

EC50, Reproduction

with Confidence Interval (CI) p = 95 %

94.9 mg/L

(CI: 77.1 - 117 mg/L)

SD       = Standard deviation

1)         = Statistical significance (One Way Analysis of Variance, DUNNETT’S method, p = 0.05)

2)         = First brood significantly delayed (later than day 9) at seven of ten parent animals

3)         = Number of broods significantly reduced at seven of ten parent animals

---         = Not applicable due to 100 % mortality of the parent animals in the course of the study

Test Item Related Effects on Adult Mortality and Growth (Weight, Length) and EC50, Adult Mortality

Effects

Nominal Concentration [mg/L]

Control

25.0

50.0

100

200

400

Adult mortality after 21 days [%]

0

0

0

0

1001)

1001)

Parent animals: dry weight [mg]

0.89

0.97

0.85

0.572)

---

---

Parent animals: total length [mm]

5.30

5.25

4.882)

4.202)

---

---

EC50, Adult Mortality

with Confidence Interval (CI) p = 95 %

141 mg/L

(CI: 100 - 200 mg/L)

1)  =  Mortality > 20 % biologically significant

2)  =  Growth of the parent animals significantly reduced in comparison to the control group

--- =  not applicable due to 100 % mortality of the parent animals before test end

Stillborn Juveniles and Aborted Eggs after 21 Days

Nominal
concentration

Number of

Total No.

Mean Stillborn Juveniles and Aborted Eggs per Parent alive

Stillborn

Aborted

[mg/L]

Juveniles

Eggs

å

N

MV ± SD

400

--

--

--

  0

--

200

--

--

--

  0

--

100

8

3

11

10

1.10 ± 1.29

    50.0

8

5

13

10

1.30 ± 1.42

    25.0

0

2

  2

10

0.20 ± 0.42

Control

0

0

  0

10

0 ± 0

MV ± SD = Mean value ± Standard deviation

--  = Not applicable, due to mortality of all parent animals

N = Number of parents producing juveniles alive

Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles

Nominal
Test Item
Concentration

Number of Juveniles

Percentage of Dead Juveniles *
[%]

[mg/L]

Dead

Alive

Total

400

--

--

--

--

200

--

--

--

--

100

11

  559

  570

2

    50.0

13

1051

1064

1

    25.0

  2

1262

1264

0

Control

  0

1210

1210

0

Dead    = Aborted eggs + stillborn juveniles

Total    = Dead + alive juveniles

*)         = Related to the total number of juveniles

--         = Not applicable, due to mortality of all parent animals

Intrinsic Rates of Natural Increase

Nominal
concentration

Intrinsic Rate of Natural Increase in Replicate No.

Mean IR

CV

[mg/L]

1

2

3

4

5

6

7

8

9

10

MV ± SD

[%]

400

--

--

--

--

--

--

--

--

--

--

--

--

--

200

--

--

--

--

--

--

--

--

--

--

--

--

--

100

0.46

0.41

0.41

0.41

0.41

0.39

0.36

0.45

0.41

0.45

0.42* ±

0.03

8

50.0

0.48

0.48

0.52

0.50

0.48

0.51

0.51

0.49

0.47

0.50

0.49  ±

0.02

3

25.0

0.49

0.51

0.49

0.51

0.53

0.53

0.50

0.52

0.51

0.51

0.51  ±

0.01

3

Control

0.53

0.50

0.49

0.49

0.49

0.49

0.52

0.50

0.52

0.50

0.50  ±

0.01

3

IR        = Intrinsic rate of natural increase

MV ± SD        = Mean value ± Standard deviation

CV      = Coefficient of variation

--         = Not applicable, due to mortality of all parent animals

*)         = Statistical significance (DUNNETT’S method, p = 0.05)

First Appearance of Living Juveniles in the Individual Groups

Nominal
concentration

Day of First Appearance of Living Juveniles

First

in Replicate No.

Appearance

[mg/L]

1

2

3

4

5

6

7

8

9

10

Mean Day

400

--

--

--

--

--

--

--

--

--

--

--

200

--

--

--

--

--

--

--

--

--

--

--

100

8

10*

10*

10*

10*

10*

10*

8

10*

8

  9.4*

    50.0

8

 8

 8

 8

 8

 8

 8

8

 8

8

8.0

    25.0

8

 8

 8

 8

 8

 8

 8

8

 8

8

8.0

Control

8

 8

 8

 8

 8

 8

 8

8

 8

8

8.0

--         = Not applicable, due to mortality of all parent animals

*)         = First brood biologically significantly delayed (later than day 9) at seven of ten parent animals

Validity criteria fulfilled:
yes
Remarks:
Mortality of the parent animal in the control group did not exceed 20 % at the end of the test. The mean number of live offspring produced per parent animal surviving at the end of the test was 121 in the control group.
Conclusions:
The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) for the reproductive output as most sensitive
effect were determined directly from the results. The EC50-value for the reproductive output was calculated by sigmoidal dose-response regression. All effect values are given based on the nominal test item concentrations.

NOECReproduction :25.0 mg/L
LOECReproduction :50.0 mg/L
EC50, Reproduction :94.9 mg/L (CI: 77.1 - 117 mg/L)
Executive summary:

The 21-day-chronic toxicity of Dimethylaminopropyl methacrylamide to Daphnia magna was studied under static renewal conditions.  Daphnids were exposed to control and test chemical at nominal concentrations of 25, 50, 100, 200 and 400 mg /L. The 21 day EC50 based on reproduction effect was 94.9 mg /L. The 21-day NOEC based on reproduction was 25.0 mg/L. The sublethal effects included were occurence of aborted eggs and stillborn juveniles, time of production of first brood, adult mortality, intrinsic rate of natural increase, body weight and length of the parents. 

This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates. 

Results Synopsis 

Test Organism: Daphnia magna:

Test Type: Static Renewal

NOEC:  25.0 mg /L

LOEC:  50.0 mg /L                   

Endpoint Effected:  reproduction

Description of key information

The long term toxicity of the substance to Daphnia magna has been evaluated in a 21 day static reproductive toxicity test according to 
OECD guideline 211. The 21-day NOEC was 25 mg/l, based on reproduction. (DR.U.NOACK-LABORATORIEN, 2009)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
25 mg/L

Additional information

There is one long-term ecotoxicity study in freshwater invertebrates available for Dimethylaminopropyl methacrylamide.

The long term toxicity of the substance to Daphnia magna has been evaluated in a 21 day static reproductive toxicity test according to OECD guideline 211.

Daphnids were exposed to control and test chemical at nominal concentrations of 25, 50, 100, 200 and 400 mg/L.

The 21-day NOEC was 25 mg/l (based on reproduction). (DR.U.NOACK-LABORATORIEN, 2009)