Reaction products of Soya and Linseed Oil Fatty Acids with Bisphenol A diglycidylether

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:

12 February 2019 - 29 June 2019 (experimental end)

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)

Version / remarks:

Adopted April 13, 2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the controls according to the schedule below. Stock solutions in ACN used for preparation of test solutions were sampled as well.

Frequency t=0 h and t=48 h
Volume 2.0 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.

Vehicle:

yes

Remarks:

However the vehicle was evaporated away prior to dissoution into media.

Details on test solutions:

Based on the results obtained in the preceding tests, Water Accommodated Fractions (WAFs) were prepared by using stock solutions in ACN with nominal concentrations of 0.050, 0.075, 0.11, 0.17 and 0.25 mg/mL in the first final test and 0.011, 0.024, 0.050, 0.12 and 0.25 mg/mL in the second final test. Empty test vessels were individually spiked with 1.0 mL of the respective stock. Each replicate of the solvent control received 1.0 mL ACN. The solvent was completely evaporated overnight before addition of the test medium to avoid modification of the WAF-composition due to presence of a water-miscible solvent. Thereafter, 50 mL test medium was added to each vessel. Test solutions, including those of the blank control and the solvent control, were agitated for a period of two days to ensure maximum dissolution of test item in test medium. Vessels were sealed during agitation to prevent evaporation of test medium. All test solutions were clear and colourless at the end of the preparation procedure.

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 culture with a known history.
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 Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Breeding
Start of each batch Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed 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.
Culture 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:

The hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 19 and 21°C during the test, and complied with the requirements as laid down in the study plan (18 22°C, constant within ±1°C).

pH:

The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 6. These test conditions remained within the limits prescribed by the study plan (pH: 6 9, not varying by more than 1.5 units; oxygen: 3 mg/L at the end of the test).

Dissolved oxygen:

The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 6. These test conditions remained within the limits prescribed by the study plan (pH: 6 9, not varying by more than 1.5 units; oxygen: 3 mg/L at the end of the test).

Nominal and measured concentrations:

Time of sampling Date of sampling Date of analysis1 Loading rate 2 (mg/L) Concentration analyzed (mg/L)
0 17-Jun-19 28-Jun-19 0 n.d.
0 3 n.d.
0.21 n.d.
0.47 n.d.
1 n.d.
2.3 0.00030 4
5 0.00041 4
48 19-Jun-19 28-Jun-19 0 n.d.
0 3 n.d.
0.21 n.d.
0.47 n.d.
1 n.d.
2.3 0.000095 4
5 0.00013 4
1 Samples were stored in the freezer (≤ -15°C) until the day of analysis.
2 A water accommodated fraction (WAF) prepared at the loading rate.
3 Solvent control.
4 Estimated value, calculated by extrapolation of the calibration curve.
n.d. Not detected. The limit of detection of the analytical method was determined to be 0.23 µg/L taking a dilution factor of two into account.

Details on test conditions:

Test Procedure and Conditions
Test duration; 48 hours
Test type Static
Test vessels 100 mL, all-glass
Test medium Adjusted ISO medium. The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
The hardness of test medium expressed as CaCO3: 180 mg/L.

Number of daphnids 20 per test concentration and control
Loading 5 per vessel containing 50 mL of test solution.
Light A daily photoperiod of 16 hours.
Feeding No feeding
Aeration No aeration of the test solutions was applied.
Introduction of daphnids Within 51 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading weight

Basis for effect:

mobility

Details on results:

No statistically significant differences were determined for the blank and the solvent control groups. The data recorded for both groups were thus pooled for the subsequent determination of effect parameters. No immobility was observed in the controls or the two lowest test concentrations throughout the exposure period. At the end of the test, a dose-related increase of immobility was observed at WAFs individually prepared at a loading rate of 1.0 mg/L and higher, reaching 100% immobility at the highest test concentration.

Results with reference substance (positive control):

The study met the acceptability criteria prescribed by the study plan and was considered valid.
The 24h-EC50 in this reference test with K2Cr2O7 was within the expected range of 0.6 and 2.1 mg/L as specified in ISO International Standard 6341, October 2012. The 48h-EC50 was within the expected range of 0.28 and 0.75 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
The 24h-EC50 was 0.92 mg/L with a 95% confidence interval between 0.78 and 1.1 mg/L.
The 48h-EC50 was 0.55 mg/L with a 95% confidence interval between 0.47 and 0.64 mg/L.
In conclusion, the sensitivity of this batch of Daphnia magna was in agreement with ISO International Standard 6341, October 2012 and the historical data collected at Charles River Den Bosch.

Reported statistics and error estimates:

Parameters of the Probit Analysis at 48h

Parameter Value
Computation runs: 8.00000
Slope b: 4.87127
Intercept a: -0.23735
Variance of b: 0.88360
Goodness of Fit
Chi²: 1.88047
Degrees of freedom: 3.00000
p(Chi²): 0.59800
Log LC50: 0.04872
SE Log LC50: 0.04446
g-Criterion: 0.14304
F: 42.84400
p(F) (df: 1;3): 0.00700

Results of the Probit Analysis at 48h

Parameter EL50
Value [mg/L] 1.119
lower 95%-cl 0.915
upper 95%-cl 1.367

Time (h)	Replicate	Soya/Linseed Oil Fatty Acid-BADGE reaction product; WAF (mg/L)
		Blank control	Solvent control	0.21	0.47	1.0	2.3	5.0
0	A	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20
24	A	0	0	0	0	0	0	2
	B	0	0	0	0	0	1	1
	C	0	0	0	0	0	1	3
	D	0	0	0	0	0	0	3^#
	Total immobilised	0	0	0	0	0	2	9
	Effect %	0	0	0	0	0	10	45
48	A	0	0	0	0	2	5	5
	B	0	0	0	0	1	5	5
	C	0	0	0	0	4	4	5
	D	0	0	0	0	3	4	5
	Total immobilised	0	0	0	0	10	18	20
	Effect %	0	0	0	0	50	90	100

^#Microscopic observation revealed no test item attached to the daphnids.

Validity criteria fulfilled:

yes

Conclusions:

the 48h-EL50 for Daphnia magna exposed to Soya/Linseed Oil Fatty Acid BADGE reaction product was 1.1 mg/L (95% confidence interval between 0.92 and 1.4 mg/L).

Executive summary:

The objective of the study was to evaluate Soya/Linseed Oil Fatty Acid-BADGE reaction product for its ability to generate acute toxic effects on the mobility ofDaphnia magnaduring an exposure period of 48 hours and, if possible, to determine the EL₅₀at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004.In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23,2019.

The batch of Soya/Linseed Oil Fatty Acid-BADGE reaction product tested was a clear colourless viscous liquid UVCB and not completely soluble in test medium at the loading rates initially prepared.Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 0.21 and 5.0 mg/L and used as test concentrations. Test solutions were prepared by using stock solutions in acetonitrile (ACN). Since the test item is a UVCB, the solvent was completely evaporated before addition of test medium to avoid modification of the WAF-composition due to presence of a water-miscible solvent.

A full test was performed based on the results of a preceding combined limit/range-finding test, solubility trials and a first full test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to a blank control, a solvent control and to WAFs individually prepared at loading rates of 0.21, 0.47, 1.0, 2.3 and 5.0 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken from the test solutions at the start and at the end of the test,as well as from the stock solutions used in the preparation of the test solutions.

Samples taken from the stock solutions of Soya/Linseed Oil Fatty Acid-BADGE reaction product in ACN used in the preparation of test solutions were analysed. The analysed concentrations were at 106-121% relative to the nominal concentrations, indicating proper preparation of the stocks. In addition,samples taken from all test concentrations and the controls were analysed at the start and at the end of test. The measured concentrations were either not detected or calculated by extrapolation of the calibration curve throughout the test.

Based on these results and considering that the test item is a UVCB, the effect parameters were based on the nominal loading rates.Indicative effect parameters based on detected concentrations could not be determined.

No immobility was observed in the controls and the two lowest test concentrations throughout the exposure period. At the end of the test, a dose-related increase of immobility was observed at WAFs individually prepared at a loading rate of 1.0 mg/L and higher, reaching 100% immobility at the highest test concentration.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EL₅₀forDaphnia magnaexposed to Soya/Linseed Oil Fatty Acid‑BADGE reaction product was 1.1 mg/L (95% confidence interval between 0.92 and 1.4 mg/L).

Description of key information

48 h EL50 = 1.1 mg/L (OECD TG 202, Daphnia magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.1 mg/L

Additional information

The acute toxicity of Soya/Linseed Oil Fatty Acid-BADGE reaction product to Dapnia magna was determined in a study according to OECD TG 202 (2004)

Soya/Linseed Oil Fatty Acid-BADGE reaction product was a clear colourless viscous liquid UVCB and not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 0.21 and 5.0 mg/L and used as test concentrations. Test solutions were prepared by using stock solutions in acetonitrile (ACN). Since the test item is a UVCB, the solvent was completely evaporated before addition of test medium to avoid modification of the WAF-composition due to presence of a water-miscible solvent.

A full test was performed based on the results of a preceding combined limit/range-finding test, solubility trials and a first full test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to a blank control, a solvent control and to WAFs individually prepared at loading rates of 0.21, 0.47, 1.0, 2.3 and 5.0 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken from the test solutions at the start and at the end of the test, as well as from the stock solutions used in the preparation of the test solutions.

Samples taken from the stock solutions of the test item in ACN used in the preparation of test solutions were analysed. The analysed concentrations were at 106-121% relative to the nominal concentrations, indicating proper preparation of the stocks. In addition, samples taken from all test concentrations and the controls were analysed at the start and at the end of test. The measured concentrations were either not detected or calculated by extrapolation of the calibration curve throughout the test.

Based on these results and considering that the test item is a UVCB, the effect parameters were based on the nominal loading rates. Indicative effect parameters based on detected concentrations could not be determined.

No immobility was observed in the controls and the two lowest test concentrations throughout the exposure period. At the end of the test, a dose-related increase of immobility was observed at WAFs individually prepared at a loading rate of 1.0 mg/L and higher, reaching 100% immobility at the highest test concentration.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EL50 for Daphnia magna exposed to Soya/Linseed Oil Fatty Acid‑BADGE reaction product was 1.1 mg/L (95% confidence interval between 0.92 and 1.4 mg/L).

 

Supporting data are also available for the source substance BADGE, which is a main constituent of the target substance Soya/Linseed Oil Fatty Acid-BADGE reaction product.

Three studies reported 48-hour EC50 values ranging from 1.1 to 2.8 mg/L and one study reported a 48-hour EC50 value of 51.8 mg/L BADGE. Since one EC50 value was significantly greater than the other values, this data point was not factored into the calculation of a geometric mean for determining the 48-hour EC50 value for D. magna. This was in accordance with ECHA guidance (R.10.2.2) which states that if toxicity values are more than one order of magnitude different for the same species, it is questionable whether the data point should be incorporated together into the geometric mean calculation for that species and endpoint. In this case, although not reported in the study, it is probable that the test material was added to the test vessels at concentrations well above its water solubility, therefore the EC50 value of 51.8 mg/L BADGE was not incorporated into the geometric mean calculation. Thus, the key parameter for the freshwater invertebrate studies was the EC50 value of 1.8 mg/L, which was the geometric mean of five 48-hour EC50 values (1.1, 1.4, 1.7, 2.7, and 2.8 mg/L).