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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:
15 September 2017 - 27 October 2017
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:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Version / remarks:
ISO International Standard 6341.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Frequency: at t=0 h and t=48 h
- Volume: 2.0 mL from the approximate centre of the test vessels
- Storage conditions before the analysis: amples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Note:
-At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Additionally, reserve samples of 2.0 mL were taken for possible analysis.
Vehicle:
no
Details on test solutions:
PREPARATION OF THE TEST SOLUTION
- The batch of the test substance tested was a colourless to light yellow viscous liquid with a purity of 97%.
- The test item appeared not completely soluble in test medium at the concentration prepared. No correction was made for the purity/composition of the test item.
- Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to accelerate and ensure maximum dissolution of the test item in medium. The obtained hazy mixture was allowed to settle for approximately one hour. The observed undissolved fraction did not settle during this period and a test collection by siphoning over glass wool did not yield any improvement. The obtained hazy mixture was thus used directly as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The undiluted saturated solution was hazy at the end of the preparation procedure while lower test concentrations were clear and colorless at the end of the preparation procedure
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: in-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Age at study initiation: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old

BREEDING
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider
(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
Final test: 19 and 21°C
pH:
Final test: 7.7 - 8.0
Dissolved oxygen:
Final test: 8.7 - 9.1 mg/L
Nominal and measured concentrations:
The test substance: Solutions containing 1.0, 10 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.
Controls: Test medium without test item or other additives
Details on test conditions:
Test duration: 48 hours
Test type: Static

MEDIUM
Medium: adjusted ISO medium, M7 as prescribed by Dr. Elendt-Schneider
Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts: CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements: B 0.125 mg/L
Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rb and Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025 mg/L
Se 0.0010 mg/L
V 0.0003 mg/L
Na2EDTA.2H2O 2.5 mg/L
Macro nutrients: Na2SiO3.9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L
The hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3.

TEST SYSTEM
Test vessels: 60 mL, all-glass
Number of daphnids: 20 each in the control and the highest test concentration: 10 each per intermediate test concentration.
Loading: 5 per vessel containing 50 mL of test solution
Introduction of daphnids: Within 14 minutes after preparation of the test solutions.

OTHER TEST CONDITIONS
Light: 6 hours photoperiod daily
Feeding: No feeding
Aeration: no aeration of the test solutions.

MEASUREMENTS AND RECORDINGS:
Immobility (including mortality): At 24 hours and at 48 hours.
Dissolved oxygen & pH: At the beginning and at the end of the test, for the highest test concentration and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

TEST CONCENTRATIONS
The test substance: Solutions containing 1.0, 10 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.
Controls: Test medium without test item or other additives.

Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 52 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
Measured concentration of peak 2 (see details on results)
Basis for effect:
mobility
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 52 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
Measured concentration of peak 2 (see details on results)
Basis for effect:
mobility
Details on results:
EFFECTS:
No effects were observed in the control or any of the test concentrations after 24 hours of exposure. At the end of the test, no immobility was observed in the control and the two lower test concentration, while 40% of daphnids were immobile at the highest test concentration.

CONCENTRATION TEST ITEM AND RESULTS
Samples taken from the control and the undiluted saturated solution were analysed. The chemical analysis was based on the two major peaks present in the chromatograms, further referred to as peak 1 and peak 2. Concentrations measured for peak 1 were stable and in agreement with the expected nominal concentrations throughout the test duration. The concentrations for peak 2 were at about 52% relative to what was expected based on the loading rate throughout the test duration. Based on these results, it was decided to use the measured concentrations derived from peak 2 for the determination of effect parameters as a worst case assumption.

ANALYSIS
The 24 and the 48h-EC50 could not be determined because the observed effects were below 50%.
Results with reference substance (positive control):
The actual responses in the reference test with the positive control K2Cr2O7 were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
The 24h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.68 mg/L.
The 48h-EC50 was 0.41 mg/L with a 95% confidence interval between 0.34 and 0.46 mg/L.

TABLE: Number of Introduced Daphnids and Incidence of Immobility in the Test

Time (h)

Replicate

the test substance;% SS prep. at 100 mg/L

Control

1.0

10

100

0

A

5

5

5

5

B

5

5

5

5

C

5

 

 

5

D

5

 

 

5

Total introduced

20

10

10

20

24

A

0

0

0

0

B

0

0

0

0

C

0

 

 

0

D

0

 

 

0

Total immobilised

0

0

0

0

Effect %

0

0

0

0

 

 

 

 

48

A

0 (1)

0

0

0

B

0 (1)

0

0

2

C

0

 

 

3#

D

0

 

 

2

Total immobilised

0

0

0

8

Effect %

0

0

0

40

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.#Microscopic observation revealed no test item stuck to the daphnids

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, the 48h-EC50 for Daphnia magna exposed to the test substance was beyond the range tested, i.e. exceeded a measured concentration of 52 mg/L.
The 48h-EC50 was based on the measured concentrations derived from peak 2 as a worst case assumption.
The 24 and the 48h-EC50 could not be determined because the observed effects were below 50%.
Executive summary:

The acute toxicity of the test substance to waterfleas (Daphnia magna) under static conditions was performed according to OECD Guideline 202.

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to the undiluted saturated solution (100 mg/L), in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to solutions containing 1.0 and 10% of the saturated solution in the combined range-finding test.

The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

No effects were observed in the control or any of the test concentrations after 24 hours of exposure. At the end of the test, no immobility was observed in the control and the two lower test concentration, while 40% of daphnids were immobile at the highest test concentration.

The 48h-EC50for Daphnia magna exposed to the test substance was beyond the range tested, i.e. exceeded a measured concentration of 52 mg/L

All the criteria for acceptability of the test were met and the present toxicity study is classified as reliable without restriction according to the OECD guideline No. 202

Description of key information

A short term toxicity study in Daphnia magna with the test substance was performed according to OECD guideline No. 202 and GLP principles. The test was performed under static conditions and the total exposure period was 48 hours.

At the end of the test, no immobility was observed in the control and the two lower test concentrations, while 40% of daphnids were immobile at the highest test concentration.

The 48h-EC50for Daphnia magna exposed to the test substance was beyond the range tested, i.e. exceeded a measured concentration of 52 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
52 mg/L

Additional information

The acute toxicity of the test substance to waterfleas (Daphnia magna) was investigated under static conditions according to OECD Guideline 202.

A combined limit/range-finding test was performed:

- Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to 0 (untreated control) and 100 mg/L, in a limit test.

- In addition ten daphnids per group (5 per replicate, duplicate) were exposed to 1.0 and 10 mg/L, in the combined range-finding test.

The total exposure period was 48 hours.

No effects were observed after 24 hours of exposure. At the end of the test, no immobility was observed in the control and the two lower test concentrations, while 40% of daphnids were immobile at the highest test concentration.

The 48h-EC50for Daphnia magna exposed tothe test substance was beyond the range tested, i.e. exceeded a measured concentration of 52 mg/L

All the criteria for acceptability of the test were met and the present toxicity study is classified as reliable without restriction according to the OECD guideline No. 202