5,7-Di-t-butyl-3-[3,5-dimethyl-4-[(1,3,7,9-tetra-t-butyl-5-methyl-5H-benzo[d][1,3,2]benzodioxaphosphocin-11-yl)oxy]phenyl]-3H-benzofuran-2-one

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of the limit concentration, filtered and analyzed.

The limit concentration and the control were analytically verified via LC-MS/MS in fresh media at the start of exposure (0 hours). All samples were filtered with a disposable syringe filter before analysis. Since the measured concentration was below the lowest calibration level at the start of the exposure (0 hours), analytical monitoring at the end of the exposure was waived and the test item concentrations was considered below the lowest calibration level in the old media, too. No method validation was performed either.

Vehicle:

no

Details on test solutions:

A limit concentration of 100 mg/L of the test item was tested as a dispersion. Since the test item is sparingly soluble with a water solubility limit very likely below the analytical detection limit, a worst case approach was applied to ensure that the solubility limit under test conditions was maintained during the exposure period. Test item in excess of the water solubility limit was present in the test vessels throughout the exposure period. The limit concentration was selected based on the results of a non-GLP preliminary range finding test under static conditions, which demonstrated that the undissolved part of the test item had no adverse impact on the test organisms.
The limit concentration 100 mg/L was freshly prepared with dilution water directly in the test vessels. For this purpose, 2 mg of the test item were weighed into each test vessel and 20 mL of the dilution water were added. These dispersions were stirred with a magnetic stirrer at approximately 750 rpm for 72 hours at approximately 23 °C. The test solutions were not filtrated. Before insertion of the test organisms, the test solutions were allowed to stand for approximately 1 hour for separation of phases. Test item in excess of the water solubility limit was present in the test vessels throughout the exposure period. The test item floated on the water surface throughout the exposure period.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5).

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guidelines and is bred at the test facility.

Origin
lnstitut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH, Kathe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 hours illumination, light intensity of max. 1500 Ix

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of> 106 cells/ml. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches lnstitut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

180 mg CaCO3/L (start of exposure)

Test temperature:

20.9 deg. C (start of exposure)

pH:

Control: 7.75 (start of exposure); 7.53 - 7.99 (end of exposure)
100 mg/L: 8.11 (start of exposure); 7.54 - 7.61 (end of exposure)

Dissolved oxygen:

Control: 8.37 mg/L (start of exposure); 8.32 - 8.48 mg/L (end of exposure)
100 mg/L: 8.26 mg/L (start of exposure); 7.99 - 8.56 mg/L (end of exposure)

Salinity:

n/a

Conductivity:

460 µS/cm (start of expsoure)

Nominal and measured concentrations:

nominal: 100 mg/L
measured: < calibration (calibration range 1 - 25 µg)

Details on test conditions:

Test method
The study was performed under static conditions.

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 ml capacity, loosely covered with watch glasses

Test volume
20 ml

Dilution water
Same composition as the culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids were used for the limit concentration and the control.



Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application
The daphnids were inserted with a small amount of dilution water by pipette into the water body below the water surface.

Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 Ix

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.



Reference Test

A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility.

Reference item
Potassium dichromate p.a. (SIGMA)

Purity
99.0%

Batch number
MKBV0900V

Storage stability
2021-11-25

Test concentrations
1.00 - 2.00 - 4.00 mg/L

Ranges of validity
EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5),
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)

Test duration
2017-06-01 to 2017-06-02

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a. (SIGMA)

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004), which is equivalent to the Commission Regulation (EC) No.440/2008 Method C.2 (2008), from 2017-06-16 to 2017-06-21, with the definitive exposure phase from 2017-06-19 to 2017-06-21. The study was conducted as a limit test under static conditions over a period of 48 hours with a nominal concentration of 100 mg/L of the test item (tested as a dispersion). Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to the test item concentration and the control. Since the test item is sparingly soluble with a water solubility limit very likely below the analytical detection limit, a worst-case approach was applied to ensure that the solubility limit under test conditions was maintained during the exposure period. 2 mg of the test item were weighed into each test vessel and 20 ml of the dilution water were added. These dispersions were stirred with a magnetic stirrer at 750 rpm for 72 hours at approximately 23 °C. The test solutions were not filtrated. Before insertion of the test organisms, the test solutions were allowed to stand for approximately 1 hour for separation of phases. Test item in excess of the water solubility limit was present in the test vessels throughout the exposure period. The test item floated on the water surface throughout the exposure period. The undissolved part of the test item had no adverse impact on the test organisms. The concentrations of the test item were analytically verified via LC-MS/MS in fresh media at the start of the exposure (0 hours) in the limit concentration and the control. Before analysis, all samples were filtered to remove undissolved test item. The measured concentration of the test item (dissolved part after filtration) was lower than the calibration range (1 - 25 μg/L). Therefore, the test item concentration in old media at the end of the exposure can be considered lower than the calibration range, too.
The validity criteria of the test guidelines were fulfilled.

The study was performed according to OECD Guideline 202 (2004), which is equivalent to the Commission Regulation (EC) No.440/2008 Method C.2 (2008). The validity criteria were fulfilled:
- In the control group, no daphnids were immobilized or showed any signs of disease or stress, e.g. discoloration or unusual behavior such as trapping on the surface of the water, during the 48-hour test period (required: not more than 10% of the daphnids in the control).
- The dissolved O2 concentration in the old media at the end of the exposure was ≥ 7.99 mg/L (required: > 3 mg/L at the test end of the exposure) in all test vessels of the limit concentration and the control.

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017-06-01 to 2017-06-02.
EC50 = 2.05 mg/L
95% confidence limits: 1.80 – 2.33 mg/L
Valid range:
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading of the test item, the 48 hour-EL50 for Daphnia magna is > 100 mg/L.

Description of key information

There is a high probability that the product is not acutely harmful to aquatic invertebrates.

Additional information

Short-term toxicity to aquatic invertebrates was tested in a study conducted according to OECD TG 202 under consideration of GLP. A limit test with a concentration of 100 mg/L (nominal) was conducted. The limit concentration was freshly prepared with dilution water directly in the test vessels. The test solutions were not filtrated. Before insertion of the test organisms, the test solutions were allowed to stand for approximately 1h for separation of phases. Test item in excess of the water solubility limit was present in the tes tvessels throught the exposure period. The test item floated on the wtaer surface throughout the exposure period and had no physical effect on the daphnids. After an exposure period of 48h the EL50 was determined as >100 mg/L. The validity criteria were fulfilled.