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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:
From November 10, 2017 to November 24, 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:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The total exposure period was 48 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure
Vehicle:
yes
Remarks:
Water (WAF)
Details on test solutions:
Test substance was a white turbid liquid (UVCB) and not completely soluble in test medium at the loading rates initially prepared. A correction was made for the water content of the test substance. A correction factor of 4.99 was used, i.e. the nominal amount to be tested was multiplied with the correction factor to determine the amount of test substance to be weighed for the test solution preparation. Concentrations are expressed as “mg UVCB/L” when referring to the tested fraction of the test substance.
Preparation of test solutions started with loading rates individually prepared at loading rates of 1.0 to 100 mg UVCB/L.
A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test substance in test medium. The obtained mixtures were allowed to stabilize overnight.
Thereafter, the aqueous Water Accommodated Fractions (WAFs) were siphoned off over glass wool and used as test solutions. The lowest test solution was clear and colorless at the end of the preparation procedure, while the two highest test concentrations were slightly hazy.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory.
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.
Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

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

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 type:
static
Water media type:
freshwater
Total exposure duration:
48 h
Hardness:
180 mg/L CaCO3
Test temperature:
20 to 21°C
pH:
8.1 to 8.3
Dissolved oxygen:
8.7 mg/L
Nominal and measured concentrations:
Nominlal concentrations: 1, 10 and 100 mg/L
Measured concentrations: 19 mg/L TOC was found in in the WAF prepared at 100 mg/L
Details on test conditions:
Test Concentrations:

Test substance: WAFs prepared at loading rates of 1.0, 10 and 100 mg/L.
Controls: Test medium without test substnace or other additives.


Test Procedure and Conditions
Test duration : 48 hours
Test type: Static
Test vessels: 60 mL, all-glass
Medium: adjusted ISO medium
Number of daphnids: Control and highest test concentration: 20 per group
Intermediate test concentrations: 10 per group
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 17 minutes after preparation of the test solutions.

Sampling and Determination of TOC Concentrations
Due to the complex nature of the test item, specific analyses were considered to be difficult to achieve and it was decided with the sponsor to analyse the Total Organic Carbon (TOC) concentration as a surrogate measure.
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h and t=48 h
Volume 60 mL
Storage Samples were stored in a refrigerator (2-8°C) until analysis.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 60 mL were taken for possible analysis. If not used, these samples were stored in a refrigerator (2-8°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Apparatus Samples were injected into a Shimadzu TOC L total organic carbon analyser combined with a Shimadzu ASI-V auto sampler (Shimadzu Kyoto, Japan). Each sample was analysed in triplicate. TOC values were determined as the Total Carbon values in the pre-treated solutions minus the measured Inorganic Carbon (TOC= TC- IC).
Calibration solution(s)
TC standard: Potassium hydrogen phthalate solution (C8H5KO4, Merck, Darmstadt, Germany).
IC standards: Sodium carbonate (Na2CO3, p.a., Merck, Darmstadt, Germany) and Sodium Bicarbonate (NaHCO3, p.a. Merck, Darmstadt, Germany).

Measurements and Recordings
Immobility (including mortality): At 24 h and at 48 h
pH and dissolved oxygen: At the beginning and at the end of the test, for the control and the highest test concentration.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
other: NOEL
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
mobility
Details on results:
The 48 h EL50 for Daphnia magna exposed to test substance was beyond the range tested, i.e. exceeded a loading rate of 100 mg/L.
Results with reference substance (positive control):
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.

Measured TOC concentrations:

Samples taken from the highest test concentration and the control were analysed. At the start of the test, 19 mg/L TOC were found in in the WAF prepared at 100 mg/L. This concentration remained stable throughout the test, i.e. was at 101% of the initial concentration at the end of the test.

Since TOC-analysis is a non-specific method, the effect parameters were determined based on loading rates.


Number of Introduced Daphnids and Incidence of Immobility

Time (h)

Replicate

Test subsrtance 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

Effect %

0

0

0

0

0

0

0

0

 

 

 

 

48

A

0

0

0

0

B

0

0

0

0

C

0

 

 

0

D

0

 

 

0

Total immobilised

Effect %

0

0

0

0

0

0

0

0


 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 48 h EL50 and NOEL were determined to be >100 mg/L (nominal).
Executive summary:

A study was conducted to determine the short-term toxicity of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol] to aquatic invertebrates, according to OECD Guideline 202, in compliance with GLP. Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0, 10 and 100 mg/L and used as test concentrations. A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to WAFs prepared at 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 h and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure. No immobility or other effects were observed in the control or at any test concentrations. Samples taken from the highest test concentration and the control were analysed. At the start of the test, 19 mg/L TOC were found in the WAF prepared at 100 mg/L. This concentration remained stable throughout the test, i.e. was at 101% of the initial concentration at the end of the test. Since TOC analysis is a non-specific method, the effect parameters were determined based on loading rates. Under the conditions of the study, the 48 h EL50 and NOEL were determined to be >100 mg/L (nominal) (Augusiak, 2018).

Description of key information

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

A study was conducted to determine the short-term toxicity of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol] to aquatic invertebrates, according to OECD Guideline 202, in compliance with GLP. Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0, 10 and 100 mg/L and used as test concentrations. A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to WAFs prepared at 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 h and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure. No immobility or other effects were observed in the control or at any test concentrations. Samples taken from the highest test concentration and the control were analysed. At the start of the test, 19 mg/L TOC were found in the WAF prepared at 100 mg/L. This concentration remained stable throughout the test, i.e. was at 101% of the initial concentration at the end of the test. Since TOC analysis is a non-specific method, the effect parameters were determined based on loading rates. Under the conditions of the study, the 48 h EL50 and NOEL were determined to be >100 mg/L (nominal) (Augusiak, 2018).