Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

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:

02 September - 22 December 2016

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)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

ECT (Date of inspection: 02 February 2015 and 09 March 2015; Signed on: 12 June 2015) CIP (Date of inspection: 13 June 2016; Signed on: 08 September 2016)

Analytical monitoring:

yes

Details on sampling:

Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations. Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. After sampling and before shipment, all samples were stored in glass bottles in the dark at a temperature of ≤18°C if not advised otherwise.

Vehicle:

yes

Remarks:

OECD medium (modified) + additional NaHCO3

Details on test solutions:

One day before the test start a stock solution was prepared by suspending 0.0604 g of the test item in 3000 mL of growth medium, resulting in a nominal loading rate of 20 mg test item/L. This stock solution was shaken overhead and thereafter stirred for 22 hours on a magnetic stirrer at room temperature in the dark. The stock solution was visually checked for undissolved test item, white particles were visible on the surface of the stock solution. Therefore, the stock solution was transferred in an additional glass beaker with a drain cock above the bottom of the beaker. After transfer the solution was allow to stand for one hour and the under part of the stock solution was drain into a glass beaker. This solution was allow to stand for an additional 15 minutes and was checked for undissolved test item. Small particles were still visible. The remaining particles were removed using a pipette and disposed. Thereafter, this stock solution was used to prepare the test solution used for the conditioning of the test vessels by direct decanting. For preparation of the test solutions used for the exposure phase, the stock solution used was carefully pipetted from the bottom phase of the glass beaker to avoid transfer of potentially remaining particles.

The stock solution was prepared once to prepare the test solutions. The volume of the stock solution was large enough to prepare the test solutions and all analytical samples and solutions used for conditioning. The vessels containing the test solutions were shaken overhead and thereafter stirred for 5 minutes on a magnetic stirrer at room temperature and placed into a temperature-controlled room for temperature adaptation.

To reduce potential adsorption process of the test item to the glass of the preparation flasks during preparation of test solutions and to the test vessels used for later exposure both materials were pre-conditioned with the corresponding test solutions.

Test organisms (species):

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

Details on test organisms:

The test system used in this study was the freshwater algae Pseudokirchneriella subcapitata. The organisms were originally supplied by Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany. The green alga Pseudokirchneriella subcapitata was chosen as a representative of freshwater algae.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.0-22.0; mean 21.62 °C

pH:

8.2-9.2

Nominal and measured concentrations:

Nominal concentrations: 0.20, 0.63, 2.00, 6.32, and 20.0 mg test item/L.
Measured geometric mean concentrations over 72h: 0.138, 0.509, 1.62, 5.00, 17.4 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL glass vials, air-tightly closed by a screw-cap with PTFE lining
- Type: closed
- Fill volume: 60±2 mL
- Initial cells density: 0.3 x10^4
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 2 (without algae) + 2 (without algae, without light)

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: OECD medium (modified) + additional NaHCO3

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod:
- Light intensity and quality: Fluorescent tubes of universal white type (Osram Lumilux L 58W/865); 71.1-84.8 µE m^-2 s^-1; mean 78.2 µE m^-2 s^-1.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr207)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.61 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 1.13 - 2.01

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 1.78-2.69

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.89 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 3.41-4.38

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.509 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limit: 1.44-1.61

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.79 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidence limit: 1.71-1.89

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.42 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidence limit: 2.26-2.67

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.509 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 1.39-2.53

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 2.21-3.40

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 4.3-5.54

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidence limit: 1.78-1.99

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidence limit: 2.11-2.34

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidence limit: 2.81-3.33

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Results with reference substance (positive control):

Standard: Growth rate ErC50 (0-72 h): 0.913 mg/L (0.886 - 0.940 mg/L; 95%-CL)
Closed-Bottle: Growth rate ErC50 (0-72 h): 0.803 mg/L (0.788 - 0.818 mg/L; 95%-CL)

These results are within the historical range of the reference test results (standard open test system) of the testing laboratory (mean ErC50 = 1.088 ± 0.364 mg/L) and within the refined range of 1.10 ± 0.48 mg/L for an international ring trial (Pattard et al 2009). Therefore, the results of these reference tests are acceptable and indicate that the algae are responding normally to toxicant stress under both normal test conditions (standard open test system) and under modified test conditions (closed bottle). This confirms that the results obtained for Ethyl Safranate, which was performed in a closed system, are expected to be consistent with that obtained in a conventional system.

Light Intensity of the Test Area

The light intensity over the incubation area was measured once during the test period at six different positions:

Minimum value: 71.1 µE m^-2s^-1

Maximum value: 84.8 µE m^-2s^-1

Mean value: 78.2 µE m^-2s^-1

Number of measurements: 6

Lower range (mean value -15%): 66.5 µE m^-2s^-1

Upper range (mean value +15%): 89.9 µE m^-2s^-1

 

The light intensity over the incubation area was within ±15% from the average light intensity as required in the guideline and the study plan.

 

pH-values of Test Solutions

Table 6.1.5/3: The pH values at the beginning (pHo) and pH values at the end (pH72) of the test.

Nominal concentration (mg/L)	pH0	pH72
Control	8.3	9.1
0.20	8.3	9.2
0.63	8.3	9.2
2.00	8.3	9.1
6.32	8.3	8.4
20.00	8.2	8.3

 

The pH-values in the control exceeded the required upper limit (pH = 9) by 0.1 pH units. This deviation has no influence on the integrity of the study results, since all cell growth related validity criteria were fulfilled and the pH drift throughout all well growing treatment levels were always below 0.9 pH units.

 

Temperature of Test Solutions

Temperature in the test media was measured automatically in one test vessel during the period of the test:

Minimum value: 21.0 °C

Maximum value: 22.0 °C

Mean value: 21.62 °C

Number of measurements: 77

The temperature was within the required range of 21-24 °C, controlled at ±2 °C, as required in the guideline and the study plan.

 

Cell number as dependent on concentration of the test item and time

Table 6.1.5/4: Cell number per mL (divided by 104) in Pseudokirchneriella subcapitata as dependent on concentration of the test item and time.

Nominal Concentrations (mg/L)		Control	0.20	0.63	2.00	6.32	20.0
0 h	Replicates	6	3	3	3	3	3
	Mean [cells]*	0.3	0.3	0.3	0.3	0.3	0.3
24h	Replicates	6	3	3	3	3	3
	Mean [cells]	0.85	0.95	0.91	0.82	0.78	0.64
	Std. Dev. [cells]	0.114	0.104	0.015	0.085	0.049	0.044
	CV %	13.40	10.94	1.68	10.42	6.35	6.81
48h	Replicates	6	3	3	3	3	3
	Mean [cells]	5.89	5.86	5.67	5.98	1.16	0.66
	Std. Dev. [cells]	0.522	0.562	0.531	0.679	0.032	0.029
	CV %	8.86	9.58	9.35	11.35	2.76	4.40
72h	Replicates	6	3	3	3	3	3
	Mean [cells]	37.24	39.14	36.08	32.44	1.10	0.51
	Std. Dev.[cells]	3.856	1.581	1.688	0.654	0.108	0.085
	CV %	10.35	4.04	4.68	2.02	9.83	16.57

Mean:arithmetic mean; Std. Dev.: standard deviation; CV: coefficient of variation

*The statistical analyses were performed using the nominal initial cell number of 0.3x10⁴cells per mL

 

The factor of biomass increase in the control cultures was 124 within the 72-hour exposure period. Therefore, the data were in the range required by the guideline and the study plan (factor of at least 16).

 

Inhibition of Yield in relation to Concentration and Time

Table 6.1.5/5: Inhibition of Yield: Yield per mL (divided by 10⁴) of Pseudokirchneriella subcapitata in relation to concentration of the test item and %Inhibition caused by the test item, after 72 hours.

Nominal concentration (mg/L)	Mean yield (cells])	Std. Dev. (cells)	n	% Inhibition	Significant difference from control (Welch-t test; p ≤ 0.05)
Control	36.94	3.856	6	0.00	-
0.20	38.84	1.581	3	-5.13	No
0.63	35.78	1.688	3	3.14	No
2.00	32.14	0.654	3	13.01	Yes
6.32	0.80	0.108	3	97.83	Yes
20.0	0.21	0.085	3	99.42	Yes

Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates;

 

The EyC50 of Pseudokirchneriella subcapitata was determined to be 3.02 mg test item/Lbased on nominal concentrations, equivalent to 2.42 mg test item/L based on geometric mean measured concentrations.

The NOEC based on nominal concentration was 0.63 mg test item/L and on geometric mean measured concentrations were 0.509 mg test item/L.

 

Inhibition of Growth Rate in Relation to Concentration and Time

Table 6.1.5/6: Inhibition of Growth Rate: Growth Rate of Pseudokirchneriella subcapitata in relation to concentration of the test item and %Inhibition caused by the test item, after 72 hours.

Nominal concentration (mg/L)	Mean growth rate (day^-1)	Std. Dev. (day^-1)	n	%Inhibition	Significant difference from control (Williams test; p ≤ 0.05)
Control	1.6056	0.03530	6	0.00	-
0.20	1.6235	0.01332	3	-1.12	No
0.63	1.5964	0.01553	3	0.58	No
2.00	1.5610	0.00674	3	2.78	Yes
6.32	0.4320	0.03352	3	73.10	Yes
20.0	0.1760	0.05553	3	89.04	Yes

Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates;

 

The ErC50 of Pseudokirchneriella subcapitata was determined to be 4.91 mg test item/L based on nominal concentrations, equivalent to 3.89 mg Ethyl Safranate/L based on geometric-mean measured concentrations.

The NOEC based on nominal concentration was 0.63 mg test item/L and on geometric-mean measured concentrations were 0.509 mg test item/L.

 

In none of the concentration levels (C0 and C1-C3, up to 2.00 mg test item/L), deformed and/or damaged algal cells were observed during microscopic inspection. At the second highest concentration level (C4, 6.32 mg test item/L) the algae cells were predominantly enlarged. At the highest concentration level (C5, 20.0 mg test item/L) no algae cells were observed.

 

 

The test item concentrations were not within 80 and 120% of nominal concentrations during the test period. The following table gives a summary of the analytical data.

Table 6.1.5/7: Summary of measured concentrations at the beginning and at the end of the test.

Conc. Level	Test period (d)	Nominal concentration (mg test item/L)	Measured concentration (mg test item/L)	Recovery of nominal concentration (%)	% of measured initial (time 0 hours) concentration (%)	Geometric mean measured concentration over 72 h (mg test item/L)
Control	0	0	n.d.	n.a.	-	-
C1	0	0.20	0.138	69	-	-
C2	0	0.63	0.502	79	-	-
C3	0	2.00	1.61	81	-	-
C4	0	6.32	4.88	77	-	-
C5	0	20.0	17.1	86	-	-
Control	3	0	n.d.	n.a.	n.a.	n.a.
C1	3	0.20	0.138	69	100	0.138
C2	3	0.63	0.516	82	103	0.509
C3	3	2.00	1.64	82	102	1.62
C4	3	6.32	5.13	81	105	5.00
C5	3	20.0	17.8	89	104	17.4

LOQ = Limit of Quantification (0.1 mg/L)

n.d.: not determined, "Limit of Detection" (<0.03 mg/L)

n.a.: not applicable

 

Samples from the test solutions were analysed to determine actual levels of the test item at start of the exposure period (time 0) and following 72 hours of exposure in comparison to nominal concentrations.

The measured concentrations ranged between 69-86% at start (0 hours), and 69-89% at end of the exposure period (72 hours), based on nominal concentrations. Therefore, the measured concentrations of the test item were not within 80-120% of the nominal concentrations throughout the test period. No decrease of test item concentration throughout the exposure period was determined. Thus, the test item concentration based on initial measured concentration remained stable within ±20% throughout the exposure period.

Since some measured concentrations were < 80% nominal, for each concentration level the geometric mean concentration was calculated using the test item concentrations measured at the start (0 hours) and the end of the exposure period (72 hours).

The following geometric mean concentrations were determined: 0.138, 0.509, 1.62, 5.00 and 17.4 mg test item/L.

 

Validity Criteria

Mean biomass increase in the control cultures: factor of at least 16 within the 72-hour test period is required: Found to be 124.

Mean coefficient of variation for section-by-section specific growth rates in the control cultures should be up to 35%:Found to be 31.1%.

Coefficient of variation of average specific growth rates during test period in replicate control cultures should be up to 7%:Found to be2.2%.

Validity criteria fulfilled:

yes

Conclusions:

The effect of Ethyl Safranate on the growth of the freshwater green algal species Pseudokirchneriella subapitata in a static 72h closed-bottle test was conducted according to guideline OECD 201. The Growth rate parameter: NOEC = 0.509, EC10 = 1.61, EC20 = 2.29, EC50 = 3.89 mg/L (mean measured) and Yield parameter: NOEC = 0.509, EC10 = 1.53, EC20 = 1.79, EC50 = 2.42 mg/L (mean measured). All the validity criteria were met.

Executive summary:

A GLP study on the Toxicity to Algae (Pseudokirchneriella subcapitata) in a Closed-Bottle System (OECD 201) was conducted for the test item.

Pseudokirchneriella subcapitata were incubated in OECD-Medium (modified) along with additional NaHCO₃ for the duration of 72 hours. Biological parameters such as Inhibition of growth in relation to control (growth rate & yield) were measured at regular intervals. Test concentrations (nominal) used were 0.20, 0.63, 2.00, 6.32, and 20.0 mg test item/L and a control. Stastistical evalutaion was conducted using Shapiro-Wilk's Test on Normal Distribution; Levene's Test on Variance Homogeneity; Williams Multiple Sequential t-test for homogeneous variances; Weibull and Probit analysis.

The biological phase of the study was conducted at ECT Oekotoxikologie GmbH, Flörsheim am Main, Germany. Samples of test solutions were analysed at CIP — Chemisches Institut Pforzheim GmbH, Pforzheim, Germany under the responsibility of the principal investigator for analysis.

Samples from the test solutions were analysed to determine actual levels of the test item at start of the exposure period (time 0) and following 72 hours of exposure in comparison to nominal concentrations. The measured concentrations ranged between 69-86% at start (0 hours), and 69-89% at end of the exposure period (72 hours), based on nominal concentrations. Therefore, the measured concentrations of the test item were not within 80- 120% of the nominal concentrations throughout the test period. No decrease of test item concentration throughout the exposure period was determined. Thus, the test item concentration based on initial measured concentration remained stable within ±20% throughout the exposure period. Since all concentration levels were measured, the biological results are additionally calculated and reported based on geometric mean measured concentrations at each concentration level.

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period. The 72 h ErC50 based on nominal concentrations was 4.91 mg/L (95% confidence limit of 4.30 to 5.54 mg/l). The 72 h ErC10 based on nominal concentrations was 2.01 mg/L (95% confidence limit of 1.39 to 2.53 mg/l). The 72 h ErC50 based on mean measured concentrations was 3.89 mg/L (95% confidence limit of 3.41 to 4.38 mg/L). The 72 h ErC10 based on mean measured concentrations was 1.61 mg/L (95% confidence limit of 1.13 to 2.01 mg/L).

All validity criteria were fulfilled as required by the study plan as well as guideline.

Description of key information

A GLP study on the Toxicity to Algae (Pseudokirchneriella subcapitata) in a Closed-Bottle System (OECD 201) was conducted for the test item.

Pseudokirchneriella subcapitata were incubated in OECD-Medium (modified) along with additional NaHCO3 for the duration of 72 hours. Biological parameters such as Inhibition of growth in relation to control (growth rate & yield) were measured at regular intervals. Test concentrations (nominal) used were 0.20, 0.63, 2.00, 6.32, and 20.0 mg test item/L and a control.

The test item concentration based on initial measured concentration remained stable within ±20% throughout the exposure period. Since all concentration levels were measured, the biological results are additionally calculated and reported based on geometric mean measured concentrations at each concentration level.

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period. The 72 h ErC50 based on nominal concentrations was 4.91 mg/L (95% confidence limit of 4.30 to 5.54 mg/l). The 72 h ErC10 based on nominal concentrations was 2.01 mg/L (95% confidence limit of 1.39 to 2.53 mg/l). The 72 h ErC50 based on mean measured concentrations was 3.89 mg/L (95% confidence limit of 3.41 to 4.38 mg/L). The 72 h ErC10 based on mean measured concentrations was 1.61 mg/L (95% confidence limit of 1.13 to 2.01 mg/L).

Key value for chemical safety assessment

EC50 for freshwater algae:

3.89 mg/L

EC10 or NOEC for freshwater algae:

1.61 mg/L

Additional information

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for classification purposes.