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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition 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:

The alga was exposed to the test item at the nominal test concentrations of 2,
5.6, 15.7, 44 and 123.2 mg/L (respective mean measured concentrations were
0.80, 0.74, 1.31, 2.61 and 20.67 mg/L) along with a negative control and
vehicle control. Six replicates were maintained for the negative control and
vehicle control; and three replicates for the test concentrations.

Vehicle:

yes

Details on test solutions:

Test medium was algal medium as described in the OECD test guideline 201
(OECD 2006) and the preparation details are given as below.
The test medium was prepared by mixing 160 mL stock solution 1 and 16 mL
each stock solution 2, 3 and 4 with 8 L of sterilized water and then making up
the volume to 16 L using sterilized water. The pH of the medium was 7.82
during definitive test. This medium was equilibrated with carbon dioxide by
keeping it in laminar hood for two hours.

Further details can be found in the attached report and appendix 1 and 2.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

American Type Culture Collection
P.O. Box 1549
Manassas, VA 20108,
USA
Culture Maintenance
Algal cells were maintained at the test facility through sub-culturing once in
5 to 10 days from the continuous culture into fresh medium in a conical flask.
These conical flasks were incubated between 21 and 25°C (±2°C) under a
white fluorescent continuous illumination of 8000 ± 1600 Lux.

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Following parameters were observed and recorded during both range finding
test and definitive test.
• The pH of the control and the test solutions were checked at the
beginning (unused portion of test medium) and at the end of the test
(pooled test medium).
• Light intensity inside the algal chamber (at four random locations) was
measured once daily using a Lux meter.
• Maximum and minimum temperature inside the algal chamber (in the
surrogate) was recorded once daily.
• The algal biomass in each flask was determined at 24, 48 and
72 hours after the start of the test using a hemocytometer (microscopic
observation). Algal cells from at least four squares (with each 0.1 mm3
volume) in the center of slide were counted. The arithmetic mean cell
count was multiplied with 104 factor to express the cell concentration per
mL.
G19186_AGT Page 17/97
Copy No. 2/2
• The measured cell concentrations in the test cultures and controls were
tabulated together with the concentrations of the test item and the times
of measurement.

Test temperature:

22.0 to 23.4

pH:

7.78 to 7.87

Nominal and measured concentrations:

Analysis of test concentrations was conducted using a validated analytical
method. Method validation and recovery of test item was carried out at 1 and
150 mg/L test concentration.
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
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 of 15.7, 44
and 123.2 mg/L drawn at the start of test; 2 and 5.6 mg/L drawn at the end 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 0.80, 0.74, 1.31,
2.61 and 20.67 mg/L against the nominal concentration of 2, 5.6, 15.7, 44
and 123.2 mg/L, respectively.

Details on test conditions:

Outline of the Method
Exponentially growing cultures of unicellular green alga Raphidocelis
subcapitata were exposed to various concentrations of the test item over
several generations under defined conditions. The inhibition of growth in
relation to a negative control culture was determined over a period of 72
hours.
Preparation of Inoculum Culture
To adapt and to ensure that the algae are in the exponential growth phase,
inoculum culture was prepared by inoculating Raphidocelis subcapitata of
continuous/mother culture to test medium 3 to 4 days before the start of the
test. The flasks were incubated inside the algal growth chamber under the test
conditions. The cell density of inoculum culture was 70.5 x 104 /mL during
range finding test and 121.5 x 104 /mL during definitive test.
Test Conditions
Algal cells with test concentrations were incubated in the following
conditions during range finding and the definitive test. The test containers
were plugged using cotton and randomly placed in the growth chamber. Test
containers were repositioned every day during the treatment period.

Range Finding Test
Range finding test was conducted to select the test concentrations for the
definitive test. In the range finding test the test item concentrations at 0.01,
0.1, 1, 10 and 100 mg/L was used along with negative control and vehicle
control. Each group had single replicate.
Definitive Test
The definitive test was carried out using a 2, 5.6, 15.7, 44 and 123.2 mg/L
(factor of 2.8) (respective mean measured concentrations were 0.80, 0.74,
1.31, 2.61 and 20.67 mg/L) along with negative control and vehicle control.
Preparation of Test Concentrations
To prepare test concentrations of 2 (G3), 5.6 (G4), 15.7 (G5), 44 (G6) and
123.2 (G7), 2, 5.6, 15.7, 44 and 123.2 mg test item was mixed initially with
0.1 mL tertiary butyl alcohol separately and volume was made up to 1 L in a
volumetric flask using test medium, respectively. Prepared formulation was
sonicated for 45 minutes.
After sonication of test concentration, 8.3 mL of algal cell suspension was
added to each treatment group to achieve the initial biomass of
1x104 cells/mL and was mixed thoroughly.
For negative control (G1), 8.3 mL of algal cell suspension was made up to
1 L using test medium.
For vehicle control (G2), 0.1 mL tertiary butyl alcohol was added and made
up to 1 L using test medium in a volumetric flask. 8.3 mL of algal cell
suspension was added to achieve the initial biomass of 1x104 cells/mL before
final volume make up.
Considering analytical sampling requirement all test concentrations were
repeated following above procedure and respective test concentrations were
pooled together before using it for treatment or sampling.
150 mL of prepared negative control and vehicle control was dispensed into 6
test replicates and 150 mL of each test item concentration was dispensed
separately into 3 test replicates.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.897 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.246 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.61 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.31 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Details on results:

At the end of the test (72 hours), the cell biomass in the test item solutions
decreased with the increase in test concentration.
The inhibition of growth rate was 0.44, 0.36, 10.12, 42.17 and 94.75% and
the inhibition of yield was 1.93, 1.57, 35.46, 84.56 and 99.64% at mean
measured concentrations of 0.80, 0.74, 1.31, 2.61 and 20.67 mg/L,
respectively, when compared to the vehicle control. There was 1.07% and
4.55% inhibition in the percent growth rate of algal biomass and yield in
vehicle control when compared with control group, respectively. Observed
cells were found morphologically normal in all the tested concentrations at
the end of test

TABLE 1.        Range Finding Test - Test Conditions

pH of Test Solutions

 

Group	Treatment (mg/L)	pH
		Beginning of the Test	End of the Test
G1	Negative control	7.86	7.89
G2	Vehicle control	7.89	7.92
G3	0.01	7.85	7.90
G4	0.1	7.88	7.91
G5	1	7.91	7.94
G6	10	7.93	7.97
G7	100	7.96	7.99

 

Treatment days	Temperature (°C)			Light intensity (lux)		Average ± % SD
	Maximum	Minimum	Average ± % SD
Day 1	22.9	22.0	22.7 ± 0.47	7650	7665	7646 ± 88.01
				7652	7753
Day 2	23.1	22.4		7680	7667
				7710	7590
Day 3	23.4	22.8		7560	7660
				7590	7572
Day 4	22.8	22.2		7435	7600
				7782	7765

 

TABLE 2.        Range Finding Test - Average Cell Biomass

Group	Treatment (mg/L)	Average cell counts (× 10^4cells/mL)				% Reduction at end of test
		0 hour	24 hour	48 hour	72 hour
G1	Negative control	1	5.50	17.25	64	-
G2	Vehicle control	1	5.50	16.25	60	-
G3	0.01	1	6.50	16.75	73	-4.78*
G4	0.1	1	5.50	14.50	69	-3.41*
G5	1	1	4.00	10.00	58	0.83
G6	10	1	2.75	4.50	9	46.33
G7	100	1	2.25	2.00	3	73.17

Note: Comparison is between test item with vehicle control group; *: To be considered as zero

 

Percent reduction	=	(Mean value of negative control) – (Value for the treatment group)	× 100
		Mean value of negative control

TABLE 3.        Definitive Test - Test Condition

pH of Test Solutions

Group	Treatment (mg/L)	pH
		Beginning of the Test	End of the Test
G1	Negative control	7.85	7.87
G2	Vehicle control	7.83	7.86
G3	0.8	7.79	7.83
G4	0.74	7.81	7.84
G5	1.31	7.83	7.85
G6	2.61	7.78	7.81
G7	20.67	7.81	7.84

Note: Respective nominal concentrations are 2, 5.6, 15.7, 44 and 123.2 mg/L

 

 

Temperature and Light Intensity Measurements of Algal Growth Chamber

Treatment days	Temperature (°C)			Light intensity (lux)		Average ± % SD
	Maximum	Minimum	Average ± % SD
Day 1	22.7	22.5	22.2 ± 0.18	7670	7750	7675 ± 85.7
				7680	7810
Day 2	22.3	22.1		7690	7610
				7710	7620
Day 3	22.2	22.0		7660	7690
				7650	7720
Day 4	22.3	22.1		7680	7640
				7790	7430

                                                      

                                                      

TABLE 4.        Definitive Test Results - Algal Cell Biomass

Group No.	Test Concentration-Mean Measured (mg/L)	Replicate	Average Cell Counts (x 10^4 cells/mL)				Yield (x 10^4 cells/mL) at 72 hours
			0 hour	24 hour	48 hour	72 hour
G1	Negative Control	R1	1	5.50	15.50	73.50	72.50
		R2	1	5.75	16.00	73.50	72.50
		R3	1	5.25	15.50	73.50	72.50
		R4	1	6.00	16.50	73.25	72.25
		R5	1	5.75	16.00	73.50	72.50
		R6	1	6.00	16.25	73.00	72.00
		Average	1	5.71	15.96	73.38	72.38
G2	Vehicle Control	R1	1	5.00	14.25	69.75	68.75
		R2	1	5.00	14.00	70.50	69.50
		R3	1	4.75	13.25	70.25	69.25
		R4	1	5.25	15.00	70.25	69.25
		R5	1	5.00	13.75	69.75	68.75
		R6	1	5.00	15.00	70.00	69.00
		Average	1	5.00	14.21	70.08	69.08
G3	0.80	R1	1	5.25	12.50	68.75	67.75
		R2	1	4.75	13.50	68.75	67.75
		R3	1	5.00	13.25	68.75	67.75
		Average	1	5.00	13.08	68.75	67.75
G4	0.74	R1	1	4.75	13.25	69.25	68.25
		R2	1	4.50	13.25	69.00	68.00
		R3	1	4.50	13.25	68.75	67.75
		Average	1	4.58	13.25	69.00	68.00
G5	1.31	R1	1	3.25	9.00	45.00	44.00
		R2	1	3.25	9.00	46.50	45.50
		R3	1	3.00	8.75	45.25	44.25
		Average	1	3.17	8.92	45.58	44.58
G6	2.61	R1	1	3.25	5.25	10.75	9.75
		R2	1	3.25	5.50	12.25	11.25
		R3	1	3.25	5.50	12.00	11.00
		Average	1	3.25	5.42	11.67	10.67
G7	20.67	R1	1	2.25	1.25	1.25	0.25
		R2	1	2.50	1.75	1.50	0.50
		R3	1	2.50	1.50	1.00	0.00
		Average	1	2.42	1.50	1.25	0.25

Note: Respective nominal concentrations are 2, 5.6, 15.7, 44 and 123.2 mg/L

 

TABLE 5.        Definitive Test Results - Percent Growth Rate Inhibition

Group	Test Concentration-Mean Measured (mg/L)	Average Cell Counts (x 10^4 cells/mL)		Natural log (average cell counts)		Growth rate	Growth rate Inhibition (%)
		0 hours	72 hours	0 hours	72 hours
G1	Negative Control	1	73.38	9.21034	13.50599	0.05966	-
G2	Vehicle Control	1	70.08	9.21034	13.45998	0.05902	1.07
G3	0.8	1	68.75	9.21034	13.44082	0.05876	0.44
G4	0.74	1	69.00	9.21034	13.44445	0.05881	0.36
G5	1.31	1	45.58	9.21034	13.02981	0.05305	10.12
G6	2.61	1	11.67	9.21034	11.66736	0.03413	42.17
G7	20.67	1	1.25	9.21034	9.43348	0.00310	94.75

Note: Comparison is between test item with vehicle control group;

Respective nominal concentrations are 2, 5.6, 15.7, 44 and 123.2 mg/L

 

 

Percent inhibition in average specific growth rate	=	(Mean value for average specific growth rate in the negative control) – (Average specific growth rate for the treatment replicate)	× 100
		Mean value for average specific growth rate in the negative control

 

TABLE 6.        Definitive Test Results - Percent Yield Inhibition

Group No.	Test Concentration-Mean Measured (mg/L)	Average Cell Counts (x 10^4 cells/mL)		Yield (x 10^4 cells/mL) at 72 hours	Yield Inhibition (%)
		0 hour	72 hours
G1	Negative Control	1	73.38	72.38	-
G2	Vehicle Control	1	70.08	69.08	4.55
G3	0.8	1	68.75	67.75	1.93
G4	0.74	1	69.00	68.00	1.57
G5	1.31	1	45.58	44.58	35.46
G6	2.61	1	11.67	10.67	84.56
G7	20.67	1	1.25	0.25	99.64

Note: Comparison is between test item with vehicle control group;

Respective nominal concentrations are 2, 5.6, 15.7, 44 and 123.2 mg/L

 

Percent inhibition in yield	=	(Mean value for yield in the negative control) – (Value for yield for the treatment replicate)	× 100
		Mean value for yield in the negative control

TABLE 7.        Definitive Test- Specific Growth Rate: Negative Control

Replicate	Specific Growth rate			Replicate-mean specific growth rate	S.D.	% CV
	0 h- 24 h	24 h - 48 h	48 h - 72 h
R1	1.71	1.03	1.56	1.43	0.36	24.92
R2	1.75	1.02	1.53	1.43	0.37	26.13
R3	1.66	1.08	1.56	1.43	0.31	21.63
R4	1.79	1.01	1.49	1.43	0.39	27.51
R5	1.75	1.02	1.53	1.43	0.37	26.13
R6	1.79	1.00	1.50	1.43	0.40	27.95
Mean ± SD (% CV)	1.74 ± 0.05 (2.87)	1.03 ± 0.03 (2.73)	1.53 ± 0.03 (1.92)	1.43 ± 0 (0.12)	-	25.71
Mean coefficient of variation (% CV) for section by section specific growth rates						25.71
The coefficient of variation (% CV) of average specific growth rates during the whole test period in replicate control cultures						0.12

 

TABLE 8.        Definitive Test- Specific Growth Rate: Treatment Group

Group	Test Concentration-Mean Measured (mg/L)	Replicate	Specific Growth Rate
			0 h - 24 h	24 h – 48 h	48 h - 72 h
G2	Vehicle control	R1	1.61	1.05	1.59
		R2	1.61	1.03	1.62
		R3	1.56	1.02	1.67
		R4	1.66	1.05	1.54
		R5	1.61	1.01	1.63
		R6	1.61	1.10	1.54
		Average	1.61	1.04	1.6
		SD	0.03	0.03	0.05
		% CV	1.96	3.07	3.25
G3	0.80	R1	1.66	0.87	1.7
		R2	1.56	1.04	1.63
		R3	1.61	0.97	1.65
		Average	1.61	0.96	1.66
		SD	0.05	0.09	0.04
		% CV	3.11	8.9	2.17
G4	0.74	R1	1.56	1.02	1.66
		R2	1.50	1.08	1.65
		R3	1.50	1.08	1.65
		Average	1.52	1.06	1.65
		SD	0.03	0.03	0.01
		% CV	2.28	3.27	0.35
G5	1.31	R1	1.18	1.02	1.61
		R2	1.18	1.02	1.64
		R3	1.10	1.07	1.64
		Average	1.15	1.04	1.63
		SD	0.05	0.03	0.02
		% CV	4.00	2.78	1.06
G6	2.61	R1	1.18	0.48	0.72
		R2	1.18	0.53	0.8
		R3	1.18	0.53	0.78
		Average	1.18	0.51	0.77
		SD	0	0.03	0.04
		% CV	0	5.62	5.43
G7	20.67	R1	0.81	-0.59	0
		R2	0.92	-0.36	-0.15
		R3	0.92	-0.51	-0.41
		Average	0.88	-0.49	-0.19
		SD	0.06	0.12	0.21
		% CV	7.19	-23.99	-111.13

Note: Respective nominal concentrations are 2, 5.6, 15.7, 44 and 123.2 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The end point values for growth rate and yield 72 hours are presented below
based on mean measured concentration.
At 72 hours, values given in mg/L:
ErC50 3.897
ErC20 1.8435
ErC10 1.2465
EyC50 1.765
EyC20 1.1678
EyC10 0.9412
LOEC 2.61
NOEC 1.31
Note: 1) ErC refers to growth rate; EyC refers to yield.
2) Range mentioned below EC value refers to 95 % fiducial limits.

Executive summary:

SUMMARY

The effect of Bisphenol C Epoxywas tested on the growth of freshwater unicellular green algaRaphidocelis subcapitata(formerlyPseudokirchneriella subcapitata)for 72 hours.

The alga was exposed to the test item at the nominal test concentrations of 2, 5.6, 15.7, 44 and 123.2 mg/L (respective mean measured concentrations were 0.80, 0.74, 1.31, 2.61 and 20.67 mg/L) along with a negative control and vehicle control. Six replicates were maintained for the negative control and vehicle control; and three replicates for the test concentrations. The initial cell density of algal cells at the start of exposure was 1 x 10⁴/mL. The cell growth was measured at 24, 48 and 72 hours after the initiation of the test using a hemocytometer (microscopic observation).

The concentration/effect relationship was determined using two factors namely growth rate and yield at the end of the test (72 hours). The inhibition of growth rate was 0.44, 0.36, 10.12, 42.17 and 94.75% and the inhibition of yield was 1.93, 1.57, 35.46, 84.56 and 99.64% at mean measured concentrations of 0.80, 0.74, 1.31, 2.61 and 20.67 mg/L, respectively, when compared to the vehicle control. There was 1.07% and 4.55% inhibition in the percent growth rate of algal biomass and yield in vehicle control when compared with control group, respectively. Observed cells were found morphologically normal in all the tested concentrations at the end of test.

The test item was recoverable at the concentrations of 1 and 150 mg/L in the vehicle. The active ingredient concentration analysis in all test concentrations showed that the mean per cent agreement with nominal concentration was 2.586 to 28.897% at the start of test and 7.815 to 102.450% at end of test (72 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 end point values for growth rate and yield 72 hours are presented below based on mean measured concentration.

 

At 72 hours	EC values (mg/L)
ErC50	3.897
ErC20	1.8435
ErC10	1.2465
EyC50	1.765
EyC20	1.1678
EyC10	0.9412
LOEC	2.61
NOEC	1.31

 

Note: E_rC refers to growth rate; E_yC refers to yield

Description of key information

The end point values for growth rate and yield 72 hours are presented below

based on mean measured concentration.

At 72 hours, values given in mg/L:

ErC50 3.897

ErC20 1.8435

ErC10 1.2465

EyC50 1.765

EyC20 1.1678

EyC10 0.9412

LOEC 2.61

NOEC 1.31

Note: 1) ErC refers to growth rate; EyC refers to yield.

2) Range mentioned below EC value refers to 95 % fiducial limits.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.897 mg/L

EC10 or NOEC for freshwater algae:

1.246 mg/L