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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:
2004-08-11 to 2004-09-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentrations of CHPTAC and its conversion product 2,3-Epoxypropyltrimethylammonium chloride (EPTAC) were determined via HPLC/MS MS in all test item concentrations (0.17, 0.51, 1.52, 4.56, 13.7 mg/l) and controls.

- Sampling method: Samples were taken on days 0, 7, 14, 19 (fresh media) and days 2, 9, 16, 21 (old media). 
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: a stock solution of 100 mg/L (i.e. 65.2 mg/L of the active ingredient CHPTAC) is said to be dispersed by agitation. No further details on the preparation of the test solution are reported. Given the high water solubility of the test substance it is assumed by the reviewer that either serial dilutions of the stock solution were made with test medium as dilution water, or that the test substance was added to the test medium to achieve the nominal concentrations desired.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Clone: 5

- Source: the test organisms were bred at the testing facility, origin Insitut fur Wasser-, Boden- und Lufthygiene (WaBoLu).

- Age of parental stock (mean and range, SD): 2-24 h old, the test organisms were not the first brood progeny.

- Feeding during test

- Food type and amount: The food quantity was a D. subspicatus (0.395 - 1.45 mL) and C. vulgaris (0.375- 0.745 mL) suspension of algae per test vessel (corresponding to 0.1 - 0.2 mg C per Daphnia per day). lt varied according to the growth of the daphnids, but was the same for all test groups on any particular day.

- Frequency: At lest 5 times per week.


ACCLIMATION

- Acclimation conditions: the test organisms were bread in an incubator in similar conditions to the test (temperature, illumination and photoperiod), in 2-3 L glass vessels with approx. 1.8 L culture medium.

- Type and amount of food: a mix of D. subspicatus and C. vulgaris with algae cell density of >10^6 cells/ml ad libitum.

- Feeding frequency: at least 5x per week

- Health during acclimation: the stock population is defined as healthy by the authors of the report.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
total hardness > 140 mg/L as CaCO3
Test temperature:
range: 20.3 to 21.6 degC
pH:
The initial pH values varied from 7.85 to 8.75 at 1.52 and 13.7 mg a.i./L, respectively and from 8.06 to 8.61 in the control. The final pH values varied from 7.55 to 7.73 at 1.52 mg a.i./L, from 8.4 to 8.9 at 13.7 mg a.i./L and from 7.71 and 8.58 in the control.
Dissolved oxygen:
The final oxygen concentrations of the measured replicates in the test media of nominal 1.52, 13.7 mg a.i./L and control were found to be greater than or equal to 7.64 mg/L.
Nominal and measured concentrations:
Nominal concentrations (test item): 0.26, 0.78, 2.33, 7.00, 21.0 mg/L
Nominal concentrations (active ingredient, a.i.): 0.17, 0.51, 1.52, 4.56, 13.7 mg a.i./L
Details on test conditions:
TEST SYSTEM

- Material, size, headspace, fill volume: glass beakers (5 (ID) x 8 (h) cm), 100 ml filled with 50 ml test solution.

- Renewal rate of test solution (frequency): 3 times per week

- No. of organisms per vessel: 1

- No. of vessels per concentration (replicates): 10

- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Elendt M4, according to Elendt (1990), modified to a total hardness of 160-180 mg/L as CaCO3

- Total organic carbon: 1.44 mg/L

- Particulate matter: <0.1 mg/L

- Metals: within acceptable ranges

- Pesticides: <0.025 ug/L

- Chlorine: <0.003 mg/L

- Conductivity: <0.1 uOhm

- Culture medium different from test medium: culture medium - Elendt M4, according to Elendt (1990), modified to a total hardness of 160-180 mg CaCO3/L

- Intervals of water quality measurement: Dissolved oxygen concentration, pH, total hardness and water temperature were measured once a week, in fresh and old media, in one replicate of the control and the highest concentration of the test item.


OTHER TEST CONDITIONS

- Photoperiod: 16h/8h light/dark cycle

- Light intensity: max 20 uE /m^2/s


EFFECT PARAMETERS MEASURED:

o mortality of parent daphnia - measured once per day
o newborn offspring first appearance - daily;
o number of live and dead offspring - daily;
o aborted eggs - daily;
o abnormalities of parent daphnia - each day of observation;
o body length and weight (parent daphnia) - end of test.


RANGE-FINDING STUDY

- Test concentrations: two separate range finding studies: 1.0-1000 mg/L

- Results used to determine the conditions for the definitive study: 100 and 20% immobilisation at 1000 and 100 mg/L respectively.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.51 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
1.52 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
1.52 - 4.56 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Details on results:
- Mortality of parent animals: 100% mortality occurred at 13.7 and 4.56 mg a.i./L treatments and 60% mortality at 1.52 mg a.i./L after 21 days.

- Body length and weight of parent animals: see Table 1 for details. No statistical analysis was applied to these parameters.

- Time to first brood release or time to hatch: no statistically significant difference between treatment levels and the control.

- Brood size: At 1.52 mg/l the reproduction output was significantly higher than in controls or other test groups indicating a stress reaction. No significant reduction in reproduction compared to the control was not observed at lower concentration levels.

Results with reference substance (positive control):
- Results with reference substance valid? yes

- Relevant effect levels: 24 h EC50 value of 1.94 mg/L
Reported statistics and error estimates:
The EC50 value for immobilisation of parent daphnia was calculated by probit analysis (Weber 1986) was calculated by probit analysis. While the NOEC was and LOEC for the a.i. CHPTAC for the reproduction were determined directly from the results.

Table 1: Effect of test material on growth and survival of D. magna (average per 10 replicates unless otherwise stated) during the 21 day test.

Treatment (mg a.i./L) nominal concentration

Mortality of parents (dead or immobile)

Mean no. of offspring

No. of juveniles per parent alive (mean ± SD)

Day of first appearance of juveniles

Mean length (mm)

Mean weight (mg)

No. Dead (per 10 replicates)

% (per 10 replicates)

Control

 1

 10

 970

 108 ± 5.04

7.1

5.39

1.27

13.7

 10

 100

 -

 0 ± 0

8

-

-

4.56

 10

 100

 -

 0 ± 0

8

-

-

1.52

 6

 60

 520

 130 ± 13

8

5.44

0.55

0.51

 1

 10

 960

 107 ± 11.3

7.4

5.53

1.01

0.17

 0

 0

 1063

 106 ± 8.54

7.1

5.45

1.18

- denotes when an endpoint is not applicable, e.g. the parent daphnia died before the end of the test and it was not possible to measure length and weight.

 

Table 2: Parental daphnia immobilisation results.

Endpoint

Result (mg a.i./L, nominal)

NOEC

1.03

LOEC

1.52

EC50

0.51

 

Table 3: Nominal and measured concentrations (ranges) during exposure.

 

 

Measured concentration (mg a.i./L) results

Nominal concentration

0 (control)

13.7

4.56

1.52

0.51

0.17

 Fresh media

CHPTAC

range (min.-max.)

 < LOQ

13.1

4.65-4.87

1.41-1.52

0.477-0.542

0.158-0.19

EPTAC as CHPTAC

range (min. - max.)

<LOQ

n.c.

n.c.

n.c.

n.c.

n.c.

Sum range (min. - max.)

<LOQ

13.1

4.65-4.87

1.41-1.52

0.477-0.542

0.158-0.19

 Old media (48 h)

CHPTAC

range (min.-max.)

<LOQ

8.8

3.06-3.22

1.08-1.41

0.317-0.405

0.12-0.161

EPTAC as CHPTAC

range (min. - max.)

<LOQ

3.91

1.31-1.37

0.107-0.459

0.134-0.179

0.0378-0.064

 Sum range (min.-max.)

<LOQ

12.7

4.37-4.59

1.51-1.61

0.474-0.546

0.163-0.189

n.c. defines not calculable because concentrations measured for EPTAC were <LOQ.

Measurements were only taken where living test organisms were present so for higher treatment value are not representative of the whole 21 day exposure.

Validity criteria fulfilled:
yes
Conclusions:
A 21 day NOEC value of 0.51 mg a.i./L has been determined for the effects of the test substance on reproduction of the test organism D. magna.

Description of key information

A 21 day NOEC value of 0.51 mg a.i./L has been determined for the effects of the test substance on reproduction of the test organism D. magna. The test was conducted according to the OECD guideline 211 and GLP.

Key value for chemical safety assessment

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

Additional information

The measured concentration are not within 80 % of the nominal concentrations. Given Effect concentrations are based on nominal values, following the approach in the EU Risk Assessment Report on CHPTAC (2008).