Reaction mass of meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene-2 yl)phenoxy]methyl}oxirane and 2RS)-2-({4-[2-(4-{[(2RS)-oxiran-2-yl]methoxy}phenyl)-1,1-dichloroethylidene-2-yl]phenoxy}methyl)oxirane

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:

24th April 2020 to 18th September 2020

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)

Specific details on test material used for the study:

Physical appearance : Solid
Purity as per Certificate
of Analysis
: 60 -85% (No purity correction during dose
formulation preparation)
Total Organic Content
(TOC)
: 62.3 %
Batch No. : GRM193K01
Manufactured date : 19 July 2019
Expiry date : 03 September 2021
Physico-chemical
properties
: Density: 1.32 g/cm3 (23 °C)
Storage conditions : Ambient (+15 to +25ºC)

Analytical monitoring:

yes

Details on sampling:

Stability of the test item in the test medium as well as analysis of test
concentrations was conducted using a validated analytical method. Method
validation, stability and recovery of test item was carried out at 1 and
50 mg/L test concentrations.

All the test concentrations along with the negative control and vehicle control
was analyzed for the test item concentration at the beginning and at the end
G19185_DAT Page 16/86
Copy No. 2/2
of test. For analysis, single composite sample was drawn from prepared test
concentrations and from the negative and vehicle control.
The criteria for acceptance of analysis results of test concentration were 80 to
120 % of nominal concentration with ≤ 20 % RSD of analyzed concentration.
The active ingredient concentration analysis showed that the per cent
agreement with nominal concentration of the samples of all test
concentrations analysed at the start and end of test were not within the range
of ± 20 % of the nominal concentration. The analysis of samples drawn at the
start of test was repeated using back up set. Hence, all test item
concentrations were recalculated by taking mean value from the analysis
results of start and end of the test. The calculated mean measured
concentrations are 1.30, 2.48, 8.02, 14.41 and 35.12 mg/L against the
nominal concentration of 2, 4, 8, 16 and 32 mg/L, respectively.

Vehicle:

yes

Details on test solutions:

Bisphenol C Epoxy was insoluble in plain test medium and further with aid
of vehicles such as acetone, dimethyl sulfoxide (DMSO), dimethyl
formamide (DMF) and tetrahydrofuran (THF) test item stuck to side walls or
formed undissolved particles. Following OECD Guidance document No. 23,
it was attempted to prepare water soluble fraction (WSF) and water
accommodated fraction (WAF) at various concentration of 10, 50, 100, 1000
and 2000 mg/L by keeping the preparation under low energy stirring for 24 h
and 48 h. The test item was found to stick to the magnetic stirring pellet and
concentrated at the bottom of the vessel. Further, analysis of aqueous/filtered
samples found no promising recovery of test item.
However, test item formed a suspension with settleable solids at bottom when
it was mixed with test medium containing 0.01% tertiary butyl alcohol at
100 mg/L, as per OECD Guidance Document 23, Aqueous-Phase Aquatic
Toxicity Testing of Difficult Test Chemicals. Analysis of aqueous sample
showed around 38% recovery of test item. Hence, 0.01% tertiary butyl
alcohol was selected as vehicle to prepare the required test concentrations for
the test.
Extensive solubility work was conducted to identify an appropriate dose preparation method. Details of which can be found in the attached report.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna was used as test system as it is a recommended species in the
guideline. Daphnia were sourced from cultures present at Eurofins Advinus Limited, Bengaluru, INDIA.

The test medium in which the
daphnia were maintained was changed at least twice a week and they were
fed with the unicellular green alga, Chlorella vulgaris.

One day prior to the start of the treatment, about 25 adults were incubated in
approximately 2 L of reconstituted water. The following day, the young ones
hatched which were less than 24 hour old were collected and used in the tests.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

Range Finding Test
18.6 to 19.1 oC

Definitive Test
19.1 to 19.8 oC

pH:

Range Finding Test
7.61 to 7.83

Definitive Test
7.76 to 7.80

Dissolved oxygen:

Range Finding Test
6.3 to 7.0

Definitive Test
6.4 to 6.9

Nominal and measured concentrations:

Range Finding
Neonates of daphnia were tested for immobility in test concentrations of 0.01, 0.1, 1, 10 and 100 mg/L along with negative
control and vehicle control.

Definitive Test
The definitive test was carried out using nominal test concentrations of 2, 4, 8, 16 and 32 mg/L along with negative control and vehicle control.

The calculated mean measured concentrations are 1.30, 2.48, 8.02, 14.41 and 35.12 mg/L against the nominal concentration of 2, 4, 8, 16 and 32 mg/L, respectively.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

2.48 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

There was no immobilization of daphnia in the negative control, vehicle
control and at the mean measured test concentration of 1.30 mg/L at 24 and
48 hours of exposure. The immobilization of daphnia was 10, 80, 100 and
100% at 24 h and 30, 100, 100 and 100% at 48 h exposure at mean measured
concentrations of 2.48, 8.02, 14.41 and 35.12 mg/L, respectively.
The EC50 value for Bisphenol C Epoxy at 24 hours was 4.9223 mg/L with
fiducial limits at 95 per cent ranging from 3.89098 to 6.22697 based on
mean measured concentrations.
The EC50 value for Bisphenol C Epoxy at 48 hours was 3.2289 mg/L
based on mean measured concentrations.
The LOEC (Low Observed Effect Concentration) was 2.48 mg/L based on
mean measured concentrations.
The NOEC (No Observed Effect Concentration) was 1.30 mg/L based on
mean measured concentrations.

The EC50 value for Bisphenol C Epoxy at 48 hours was 3.2289 mg/L

based on mean measured concentrations.

The LOEC (Low Observed Effect Concentration) was 2.48 mg/L based on

mean measured concentrations.

The NOEC (No Observed Effect Concentration) was 1.30 mg/L based on

mean measured concentrations.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value for Bisphenol C Epoxy at 48 hours was 3.2289 mg/L
based on mean measured concentrations.
The LOEC (Low Observed Effect Concentration) was 2.48 mg/L based on
mean measured concentrations.
The NOEC (No Observed Effect Concentration) was 1.30 mg/L based on
mean measured concentrations.

Executive summary:

The acute immobilization effect of the test item Bisphenol C Epoxy was

studied on Daphnia magna for 48 hours.

In definitive test, Daphnia magna less than 24 hours old were exposed to the

nominal concentrations of 2, 4, 8, 16 and 32 mg/L (respective mean measured

concentrations were 1.30, 2.48, 8.02, 14.41 and 35.12 mg/L) along with a

negative control and vehicle control. The number of daphnia immobilized

was recorded at 24 and 48 hours exposure.

There was no immobilization of daphnia in the negative control, vehicle

control and at the mean measured test concentration of 1.30 mg/L at 24 and

48 hours of exposure. The immobilization of daphnia was 10, 80, 100 and

100% at 24 h and 30, 100, 100 and 100% at 48 h exposure at mean measured

concentrations of 2.48, 8.02, 14.41 and 35.12 mg/L, respectively.

The stability test results concluded that the test item was stable in the test

medium at 48 h at nominal concentrations of 1 and 50 mg/L. The active

ingredient concentration analysis in all test concentrations showed that the

mean per cent agreement with nominal concentration was 52.2 to 123.275%

at the start of test and 59.338 to 119.622% at end of test (48 hour). Since the

analysis results of samples drawn at the start and end of test were out of the

acceptable limit (80 to 120 % of the nominal concentration with an RSD of <

20%) the nominal concentration of each test concentrations were recalculated

and presented as mean measured concentrations.

The EC50 value for Bisphenol C Epoxy at 48 hours was 3.2289 mg/L

based on mean measured concentrations.

The LOEC (Low Observed Effect Concentration) was 2.48 mg/L based on

mean measured concentrations.

The NOEC (No Observed Effect Concentration) was 1.30 mg/L based on

mean measured concentrations.

Description of key information

The EC50 value for Bisphenol C Epoxy at 48 hours was 3.2289 mg/L

based on mean measured concentrations.

The LOEC (Low Observed Effect Concentration) was 2.48 mg/L based on

mean measured concentrations.

The NOEC (No Observed Effect Concentration) was 1.30 mg/L based on

mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.229 mg/L