Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 october 2012 to 28 february 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
For determination of the test item concentrations, duplicate samples were taken from all test concentrations and from the control at one treatment period of the first, second and last week of the test (day 0, 7 and 16, respectively).
To determine the maintenance of the test item concentrations in the test media, stability samples were taken at the end of the two test medium renewal periods of 48 hours (days 2 and 9) and at the end of the one renewal period of 72 hours (day 19).

The samples from the highest nominal concentration of 10 mg/L were only taken on days 0 and 2 as all daphnids were dead at observation on day 2 at this concentration level.

The following stability samples were taken in duplicate :
- samples taken from the actual test by conbining the contents of all replicate test beakers after the end of the test medium renewal period
- samples incubated during the renewal periods under test conditions, however without food and daphnids.

- Sample storage conditions before analysis:
All the samples were stored deep-frozen (at about -20°C) immediiately after sampling. Based on pre-experiments for investigation of the storage stability, the test item was considered to be sufficiently stable in the test water under these storage conditions.

The concentrations of the test item were measured in one of the duplicate test media samples from the two higher nominal concentrations of 3.2 and 10 mg/L, determined in the experiment as the 21-day NOEC and LOEC. From the control samples, one of the duplicate samples of each sampling date was analyzed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: prior to the start of the test and prior to each test medium renewal, a stock solution of nominal 100 mg/L was freshly prepared by completely dissolving 50 mg of the test item (dosed in the range of 50.0 to 50.7 mg) in 500 mL of test water using stirring for 15 minutes at room temperature. Then, this intensively stirred solution was used in a series of dilution to prepare the test media of all test item concentrations.
- Controls: Yes, reconstituted water (test water)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning theappearance of the test media. All test media were clear solutions throughout the test medium renewal periods.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone : Daphnia magna Straus Clone 5
- Source: supplied by the University of Sheffield/UK in 1992. Since this date, the clone is successfully bred at Harlan Laboratories in culture medium identical to the medium used for the test.
- Age of parental stock (mean and range, SD): the daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals wer less than 24 hours old.
- Feeding during test : yes
- Food type: food mixture containing a suspension of green algae of the species Scenedesmus subspicatus (freshly grown at Harlan laboratories) and a fish food suspension (commercial fish diet Tetra Mi ®Hauptfutter).
- Amount: The food amount (based on the measured concentrations of the total Organic carbon) (TOC) in the food suspensions) was 0.20 mg TOC per Daphnia and day.
- Frequency: daily
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
water hardness : 2.5 mmol/L (=250 mg/L as CaCO3)
Test temperature:
During the test period, the water temperature was between 20 and 21°C.
pH:
During the test period, the pH of the test media and control ranged from 7.5 to 7.9.
See table 1
Dissolved oxygen:
During the test period, the dissolved oxygen concentrations were at least 7.7 mg/L.
See table 2
Salinity:
No data
Nominal and measured concentrations:
The nominal concentrations tested were 0.10, 0.32, 1.0, 3.2 and 10 mg/L (cooresponding to 0.036, 0.116, 0.36, 1.16 and 3.6 mg active ingredient/L). Additionally, a control was tested in parallel.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass-beaker
- Type : covered with glass plates
- Material, size, headspace, fill volume: 100 mL glass-beaker containing 80 mL test medium
- Aeration: Test water was aerated until oxygen saturation before the test, but not aerated during the test
- Renewal rate of test solution (frequency): Days 2, 5, 7, 9, 12, 14, 16 and 19 of the exposure period (every monday, wednesday and friday). At these dates, the surviving test animals were carefully transferred by means of glass tubes from the old test vessels into the freshly prepared test medium.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): Single
- No. of vessels per control (replicates): Single

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M7 medium). Before use, the test water was aerated until oxygen saturation. during the test, the test media were not aerated. The test water was prepared by dissolving analytical grade salts and additives (according to the OECD guidelines 211) in purified water.
- Alkalinity: 0.9 mmol/L
-Water hardness : 2.5 mmol/L (= 250 mg/L as CaCO3)

OTHER TEST CONDITIONS
- Photoperiod: A 16-h light to 8-h darkness photoperiod with a 30 minute transition period between light and darkness
- Light intensity: Approx. 400-540 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The test replicates were observed for immobility of adults on days 0 -2 and thereafter three times per week before renewal of the test media . On the same dates, the test replicates were observed for the number of living and dead offspring and for the presence of aborded eggs.
- The time weighted means were calculated using the formula mentioned in the OECD guideline 211.
- The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test.
- The mean reproduction rates of the daphnids at the test concentrations were compared to the control by Dunnett t-test (Dunnett, 1995; Dunnett, 1964).
- no EC values for the inhibition of th reproduction rate could be calculated since no effect was determined on the reproduction of the daphnids up and including the nominal test concentration of 3.2 mg/L.

CONTROL PERFORMED: Yes

RANGE-FINDING STUDY : no
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.39 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: solid content
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.05 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.39 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: solid content
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.05 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
At the concentrations of 0.10, 0.32, 1.0 and 3.2 mg/L, the survival was 100, 90, 100 and 80%, respectively (see table 3). At the highest test concentration of 10 mg/L , all test animals were dead at observation on day 5. Mortality up to 20% is regarded as natural and tolerated by the test guideline. Thus, the survival of daphnia magna after 21 days was reduced at the test concentration of nominal 10 mg/L (10.6 mg/L, 3.8 mg active ingredient/L, both mean measured).

The first young offspring released from their parent animals were recorded in the control and at the test concentrations of 0.10 to 3.2 mg/L with surviving parent animals at observation on day 9 (see table 4). At the highest concentration tested (nominal 10 mg/L), no offspring were produced.Thus the time of the first brood was not affected by the test item up and including the test concentration of nominal 3.2 mg/L (2.9 mg/L, 1.05 mg active ingredient/L, both mean measured).

The mean reproduction rate of the daphnids in the control was 127 +/- 13 living offspring per adult (mean +/- standard deviation, see table 5). No significant inhibitory effect of the test item on the mean reproduction rate was determined up to and including the highest test concentration of nominal 3.2 mg/L (Dunnett t-test, one-sided smaller, α = 0.05).

With the exception of the reported mortality and reduced reproduction rates, no visible abnormalities were observed at the test animals during the test.

1- VALIDATION OF THE ANALYTICAL METHOD

- Specificity

The biological control samples and an analyzed analytical blank (test media) did not affect the chromatogram at the retention time of the test item. The calibration solutions contained a peak specific for the test item, whose area changed accordingly with known concentration.

-Linearity

- An example of calibration data for the calibration solutions of the test item is given in figure 1 (calibration plot) and in the following table :

Nominal concentration of test item

 

(mg/L)

Signal

 

 

 

(counts)

Fitted concentration of test item

 

 

(mg/L)

Deviation of the fitted value from the nominal value

 

(%)

1.04

54353

1.01

-2.8

2.06

201412

2.02

-1.9

3.06

469701

3.16

3.2

5.00

1257400

5.31

6.2

9.54

3694335

9.39

-1.7

12.1

6168676

12.3

1.9

15.3

8485034

14.6

-4.5

The R2 fit of the calibration curves used were 0.9985, 0.9979 and 0.9985. This reflects the linearity of the analytical system within the calibration range of 1.04-15.3mg test item/L.

- Accuracy (recovery) and precision

Concurrent with the sample analyzis, a set of recovery samples accurately forftified at relevant concentrations of the test item (1.93 mg and 14.1 mg test item/L) was prepared five-fold and analyzed. The results obtained for the concentrations of the test item in the recovery samples are presented in the following table :

Nominal concentration of test item

 

 

cfort

(mg/L)

Measured concentration of test item in the Spiked sample

x

(mg/L)

Sample preparation factor

 

 

F

Concentration of test item determined in the spiked sample

c

(mg/L)

Recovery rate

 

 

 

R

(%)

Accuracy (Average Recovery)

 

 

 

(%)

Precision (Relative standard deviation of recovery)

(%)

0

*

1.0

< LOQ

n.a.

n.a.

-

1.93

2.00

1.0

2.00

104

 

 

 

105

 

 

 

5

1.93

1.97

1.0

1.97

102

1.93

2.15

1.0

2.15

112

1.93

2.06

1.0

2.06

107

1.93

1.91

1.0

1.91

99

14.1

14.9

1.0

14.9

105

 

 

110

 

 

4

14.1

15.2

1.0

15.2

107

14.1

15.3

1.0

15.3

108

14.1

15.9

1.0

15.9

112

14.1

16.2

1.0

16.2

115

Acceptance Target :

80-120

< 10

* the measured concentration was below the lowest calibration concentration

LOQ = 0.972 mg/L n.a. : not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data

The average recoveries were found to be 105% and 110% of the spiked values with relative standard deviations of 5% and 4%, respectively. The method was considered to be sufficiently accurate and precise for the purposes of this test. The test sample results were not corrected for recovery.

- Limit of quantification

The limit of quantification (LOQ) for the test item in the test samples was derived from the lowest calibration solution, which fits into the calibration curve. The LOQ is 1.04mg test item/L.

2- ANALYTICAL RESULTS

The results obtained for the concentrations of the test samples are presented in the following table :

Timepoint

 

 

 

(day/h)

Nominal concentration of test item

Cnom

(mg/L)

Measured concentration of test item

X

(mg/L)

Sample preparation factor

F

Determined concentration of test item

c

(mg/L)

% of nominal concentration

 

 

(%)

0/0

Control

**

1

< LOQ

n.a.

3.2

3.51

1

3.51

110

10

10.8

1

10.8

108

2/48

Control

**

1

< LOQ

n.a.

3.2

2.88

1

2.88

90

10

10.4

1

10.4

104

2/48*

Control

**

1

< LOQ

n.a.

3.2

1.81

1

1.81

57

10

5.21

1

5.21

52

7/0

Control

**

1

< LOQ

n.a.

3.2

3.50

1

3.50

110

9/48

Control

**

1

< LOQ

n.a.

3.2

3.44

1

3.44

108

9/48*

Control

**

1

< LOQ

n.a.

3.2

1.04

1

1.04

33

16/0

Control

**

1

< LOQ

n.a.

3.2

3.53

1

3.53

110

19/72

Control

**

1

< LOQ

n.a.

3.2

1.39

1

1.39

43

19/72*

Control

**

1

< LOQ

n.a.

3.2

1.06

1

1.06

33

* with feed ** the measured concentration was below the lowest calibration concentration LOQ = 0.972 mg/L n.a. : not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data

The concentrations as % nominal found in the fresh test samples were 110% and 108% (day 0), 110% (day 7) and 110% (day 16).

The concentrations as % nominal found in the old test samples were 90% and 104% (day 2/48h), 57% and 52% (day 2/48h, with feed), 108% (day 9/48h), 33% (day 9/48h, with feed), 43% (day 19/72h) and 33% (day 19/71h, with feed).

Remark : in the stability control samples including food particles the mean test item concentrations had further decreased to 33 -57% of the nominal values. Thus , a part of the test item had obviously adsorbed onto the food particles. However, since filter feeding test animals as Daphnia may take up the test item also from ingested food, the decrease of the test item concentrations in the test media due to adsorption onto food is not taken into account. Therefore, the biological results are based on mean measured test item concentrations (calculated as the time-weighted means over all measurements per test concentration without food (3.2 mg/L, three intervals) or as geometric mean (10 mg/L, one interval since all daphnids qere dead at day 5)).

The time weighted means (nominal 3.2 mg/L) were calculated according to the equation indicated in the OCED guidelines.

Samples with food :

Renewal

Days

Conci

mg/L

Conci+1

mg/L

ln(conci)

Ln(Conci+1)

Area

1

2

3.510

1.810

1.256

0.593

5.134

2

2

3.500

1.040

1.253

0.038

4.054

3

3

3.530

1.060

1.261

0.058

6.159

Total days : 7

 

Total area :     15.347

 

Time-weighted mean :         2,2mg/L

% of nominal :                       69%

Samples without food (used for appraisal of the biological results) :

Renewal

Days

Conci

mg/L

Conci+1

mg/L

ln(conci)

Ln(Conci+1)

Area

1

2

3.510

2.880

1.256

1.058

6.369

2

2

3.500

3.440

1.253

1.235

6.940

3

3

3.530

1.390

1.261

0.329

6.888

Total days :                7                

 

Total area :        20.198

 

Time-weighted mean :            2,9 mg/L

% of nominal :                       90%

The geometric means (nominal 10 mg/L) were calculated according to the equation indicated in the OCED guidelines.

Samples with food : 7.5 mg/L

Samples without food (used for appaisal of the biological results) : 10.6 mg/L

Validity criteria fulfilled:
yes
Conclusions:
The 21-day NOEC (survival and reproduction) was found to be 2.9 mg/L (corresponding to 1.39 mg solid content/L and 1.05 mg active content/L) based on measured concentrations. The 21-day ECx values for the inhibition of the reproduction rate could not be calculated since no effect was determined on the reproduction of the daphnids up and including the mean measured test concentration of 2.9 mg/L (corresponding to 1.39 mg solid content/L and 1.05 mg active content/L).
Executive summary:

The chronic toxicity of the test item (= substance EC 939-455-3 in aqueous commercial product) to the freshwater crustacean species Daphnia magna was investigated in a GLP-compliant study performed in accordance with OECD Guideline 211 and EU Method C.20 (Daphnia magna Reproduction Test). The 21-day NOEC (survival and reproduction) was found to be 2.9 mg/L (corresponding to 1.39 mg solid content/L and 1.05 mg active content/L) based on measured concentrations.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-457-4).
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-455-3).

3. ANALOGUE APPROACH JUSTIFICATION
Cf. attached Read-Across Justification Document (§13 Assessment reports).

4. DATA MATRIX
Cf. attached Read-Across Justification Document (§13 Assessment reports).
Reason / purpose:
read-across source
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.39 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: solid content
Basis for effect:
other: reproduction / mortality
Remarks on result:
other: read-across from EC 939-455-3
Conclusions:
By analogy with the source substance EC 939-455-3, a 21-day NOEC (survival and reproduction) of 1.39 mg solid content/L on the freshwater crustacean species Daphnia magna was reported for the target substance 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C12-18(even numbered) acyl) derivs., hydroxides, inner salts (EC 939-457-4).


Description of key information

As the chronic toxicity to aquatic invertebrates of the substance EC 939-457-4 has not been investigated experimentally, a read-across approach was followed to fill in the endpoint. The chronic toxicity to the freshwater crustacean species Daphnia magna of a structural analogue substance (= source substanceEC 939-455-3) was investigated in a GLP-compliant study (Höger, 2013) performed in accordance with OECD Guideline 211 and EU Method C.20 (Daphnia magna Reproduction Test). The 21-day NOEC (survival and reproduction) was found to be 1.39 mg solid content/L. By analogy, the same NOEC value was reported for the target substance 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C12-18(even numbered) acyl) derivs., hydroxides, inner salts (EC 939-457-4).

Key value for chemical safety assessment

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

Additional information

The chronic toxicity to aquatic invertebrates of the structural analogue substance (= source substance EC 939-455-3) was investigated in a GLP-compliant study (Höger, 2013) performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and was selected as key study for the endpoint of the target substance EC 939-457-4.