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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-13 until 2016-
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
22 Sep 2015
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were performed from the control and at the applied test concentration level at the beginning and at the end of experiment. All samples will be analysed directly after sampling.
The samples were analyzed by a HPLC method.
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION
In the main test, a stock solution with a concentration of 100 mg/L (nominal) was prepared with test item and test medium (ISO Medium). As a limit test was carried out, further dilution of stock solution was not performed.
In Preliminary range-finding test, during the formulation procedure the stock solution was prepared by the similar method described above. The test solutions were prepared by appropriate dilution of the stock solution.

Preliminary range-finding test:
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions.
Concentrations were 0.1, 1, 10 and 100 mg/L. At 100 mg/L 0% of immobilisation was observed.

Concentrations for definitive test: 100 mg/L.

Untreated control: The dilution water (ISO-medium) was used without addition of the test item.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus
- Source: István Szent University, 2100 Gödöll, Páter Károly u. 1, Hungary (Definitive test)
- Age at study initiation (mean and range, SD): They were less than 24 h old at the beginning of the test and not first brood progeny.
- Method of breeding: The Daphnia are bred in Ecotoxicological Laboratory of CiToxLAB Hungary Ltd. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
- Feeding during test: no
- Food type: The Daphnia are fed with concentrated algal suspension of Pseudokirchneriella subcapitata during the holding.


ACCLIMATION: no. There was no acclimatization because the study water used was similar to the culture water.
- Feeding frequency: The Daphnia are fed with concentrated algal suspension of Pseudokirchneriella subcapitata during the holding.
- Health during acclimation (any mortality observed): good
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
none
Hardness:
The reconstituted water (ISO medium) had an approximate theoretical total hardness of 245 mg/L (as CaCO3).
Test temperature:
The water temperature was measured at the start of the experiment and in 24-hour intervals thereafter in each test vessel. The test temperature was in the range of 20.1 20.2°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 19.6 and 20.5°C.
pH:
The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the experiment in each test vessel and was in the range of 7.46 9.03.
Dissolved oxygen:
The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the test and was in the range of 8.2 9.0 mg/L.
Salinity:
Not applicable
Nominal and measured concentrations:
Preliminary range-finding test: Nominal concentrations were tested: 0.1, 1, 10 and 100 mg/L.
Main test: Nominal concentration was tested: 100 mg/L. Measured concentration was 102.4 mg/L at the start and 107.1 mg/L at the end of the experiment.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: at least 5 mL test solution/animal
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.
No more data available.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test solution was not adjusted in the first definitive test and not varied by more than 1.5 units in any one vessel. The pH was measured at the start and at the end of the study in each test vessel and was in the range of 7.46-9.03.
- Photoperiod: 16-hour light and 8-hour dark cycle.
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.
The water temperature was measured at the start of the test and 24-hour intervals thereafter, oxygen concentrations and pH of the controls and the test solutions were measured at the beginning and at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions (in absence of stability data).
- Test concentrations: 0, 0.1, 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: There was 0% immobilisation at 100 mg/L. Because toxic response was not observed during the preliminary concentration range-finding test, a Limit Test was carried out using only one test concentration (100 mg/L) and one control group in a static system.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour (see details below).
In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Validity criteria:
No immobilization was observed in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Results with reference substance (positive control):
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 16/008-023DA) with reference item Potassium dichromate (batch no.: A0345704) is: 12 - 13 January 2016.
The 24h EC50:1.16 mg/L, (95 % confidence limits: 1.07 1.25 mg/L)
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects.
The EC50, NOEC, LOEC and EC100 values were determined directly from the raw data.

Table 1: Number and percentage of immobilised animals

Nominal concentration (mg/L)

Nb of treated animals

Immobilised animals

24 hours

48 hours

Number

percent

Number

percent

Control

20

0

0

0

0

100

20

1

5

1

5

Validity criteria fulfilled:
yes
Conclusions:
The 24h EC50 value: > 100 mg/L (nominal)
The 48h EC50 value: > 100 mg/L (nominal)
The 48h EC100 value: > 100 mg/L (nominal)
The 48h No-Observed Effect Concentration (NOEC): 100 mg/L (nominal)
The 48h Lowest Observed Effect Concentration (LOEC): > 100 mg/L (nominal)
Executive summary:

Acute toxicity of AEEU on Daphnia magna was assessed in an Acute immobilisation test, over an exposure period of 48 hours in a static test system, according to OECD 202 guideline and under GLP conditions.

Because no toxic response was observed during the preliminary range-finding test, a Limit Test was carried outusing onlyone test concentration (100 mg/L) and one control group in the definitive test. The test concentration was analytically determined at the start and at the end of the experiment. Measured concentration was 102.4 mg/L at the start and 107.1 mg/L at the end of the experiment. As the measured concentration deviated not more than 20 per cent from the nominal, biological results are based on the nominal concentration. Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentration and for the control.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of AEEU were the followings:

The 24h and 48h EC50value: >100 mg/L (nominal)

The 48h EC100value: >100 mg/L (nominal)

The 48h No-Observed Effect Concentration (NOEC):  100 mg/L (nominal)

The 48h Lowest Observed Effect Concentration (LOEC):   >100 mg/L (nominal)

All validity criteria were met during this study. 

Description of key information

The test item, AEEU, was not considered toxic to Daphnia magna at nominal concentration of 100 mg/L.

The 48-hour EC50 of AEEU to Daphnia magna was thus determined to be > 100 mg/L based on the mobility; revealing it is not harmful for the daphnids.

Key value for chemical safety assessment

Additional information

A GLP-compliant study, scored as Klimisch 1 and flagged as a key study, is available on AEEU, giving a 48h-EC50 > 100 mg/L and revealing that AEEU is not harmful to Daphnia magna (K.Sipos, 2016).

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