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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
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
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis.

Frequency at t=0 h and t=48 h
Volume 4.8 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 4.8 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤ -15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:
no
Details on test solutions:
The batch of dilithium tetraborate tested was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item. Since an undissolved material was observed, an additional 10-minute treatment of ultrasonic waves followed by 12-minute period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Stock Solution (SS) was collected by means of filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration.

Lower concentrations were prepared by diluting the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

A control which was test medium without test item or other additives was also prepared.


Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: 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: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.



Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
Not stated
Test temperature:
18-22°C, constant within 2°C
pH:
pH: 6-9, not varying by more than 1.5 units
Dissolved oxygen:
oxygen: >3 mg/L at the end of the test
Salinity:
Not stated
Conductivity:
Not stated
Nominal and measured concentrations:
A sample was taken from the undiluted stock solution and analysed.
Details on test conditions:
Test vessels: 60 mL, all-glass
Medium: Adjusted ISO medium
Number of daphnids: 20 for the control and the undiluted SS (5 per replicate, quadruplicate)
10 for the remaining test concentrations (5 per replicate, duplicate)
Loading: 5 per vessel containing 50 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids : Within 9 minutes after preparation of the test solutions.

The adjusted ISO medium consisted of the following chemicals (analytical grade) 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.

Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Samples taken from the control and the highest test concentration were analysed. Analysis of the samples showed that measured concentrations were in agreement with the nominal loading rate of 100 mg/L throughout the test period (101 – 102%). The measured concentration was at the level of nominal, despite the filtration. It was thus concluded that any undissolved material filtered was not test item related. Based on these results the effect parameters were expressed in terms of analytically confirmed nominal concentrations.

No EC50 could be calculated because the test item proved to be non-toxic at the range tested (EC50 > maximum concentration tested).

The daphnia were assessed for immobility (including mortality) at 0, 24 and 48 hours. However, no immobility or other effects were observed in the control and at any test concentrations throughout the test.

The experimental conditions (pH, temperature and oxygen concentration) were within the limits stated in the study plan.
Validity criteria fulfilled:
yes
Conclusions:
No immobility or other effects were observed in the control and at any of the test concentrations throughout the study. In conclusion, the 48h-EC50 for Daphnia magna exposed to dilithium tetraborate exceeded the analytically confirmed nominal concentration of 100 mg/L.
Executive summary:

The study was conducted to evaluate dilithium tetraborate for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50at 24 and 48 hours of exposure. The study was performed according to OECD 202.

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate) were exposed to an untreated control and to the undiluted stock solution (SS), in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to solutions containing 0.10, 1.0 and 10% of the SS 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 immobility or other effects were observed in the control and at any of the test concentrations throughout the study. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EC50 for Daphnia magna exposed to calcium metaborate exceeded the analytically confirmed nominal concentration of 100 mg/L.

Description of key information

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate) were exposed to an untreated control and to the undiluted stock solution (SS), in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to solutions containing 0.10, 1.0 and 10% of the SS 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 immobility or other effects were observed in the control and at any of the test concentrations throughout the study.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information