Ethyl 4-methyl-2-oxocyclohexanecarboxylate

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:

sept 18, 2017 to Dec 6, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 202 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Name: FRET 13-0545
Purity (Content of A.I.): 93.2%
Batch/Lot Number: PDJ378-68
Appearance: Liquid

Analytical monitoring:

yes

Details on sampling:

Water samples were collected from each treatment and control group at the beginning and end of
each renewal period during the test to determine concentrations of the test substance. Newly prepared
batch solutions were sampled on Day 0 and at 24 hours (±1 hour), and the 24-hour old solutions in each
test chamber were sampled at 24 and 48 hours (±1 hour). Samples (10.0 mL) were collected from
mid-depth, placed in Teflon tubes pre-rinsed with hexane, acidified with two drops of 10% phosphoric
acid and processed immediately for analysis.

Vehicle:

no

Details on test solutions:

A primary stock solution was prepared by mixing a calculated amount of test substance in
1000 mL of dilution water at a nominal concentration of 120 mg a.i./L, the highest concentration tested.
The stock solution concentration was adjusted to 100% active ingredient during preparation, based on the
reported test substance purity (93.2%). The stock solution was partially brought to volume and sonicated
for approximately 15 minutes. It was then brought to final volume and stirred with a Teflon®-lined stir
bar on a magnetic stir plate for approximately 15 minutes, and appeared clear and colorless with no
visible particulates. Aliquots of the primary stock solution were proportionally diluted with dilution
water to a final volume of 500 mL to prepare four additional test solutions at nominal concentrations of
7.5, 15, 30 and 60 mg a.i./L. The solutions were stirred with a Teflon®-lined stir bar on a magnetic stir
plate for approximately 15 minutes, and approximately 250 mL of solution was placed in each of two
replicate test chambers per treatment group.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnids used in the test were <24-hour old neonates obtained from cultures maintained by EAG Laboratories in Easton, Maryland.
The five adult daphnids used to supply neonates for the test were held for at least 20 days prior to collection of the juveniles for testing, and had each produced at least three previous broods.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

140 mg/L as CaCO3

Test temperature:

19.6-20.8 C

pH:

8.6-8.7

Dissolved oxygen:

≥89% ASV; no aeration

Conductivity:

405 uS/cm

Nominal and measured concentrations:

Nominal concentrations selected for use in this study were 7.5, 15, 30, 60 and 120 mg a.i./L.
Measured concentrations of the samples collected from
the newly prepared test solutions at 0 and 24 hours ranged from approximately 75.8 to 123% of nominal.
Measured concentrations of the samples collected from the 24-hour old test solutions at 24 and 48 hours
ranged from approximately 74.0 to 119% of nominal. When measured concentrations of the samples
collected during the test were averaged, the mean measured test concentrations for this study were 8.2, 13,
24, 47 and 108 mg a.i./L, representing 109, 87, 80, 79 and 90% of nominal concentrations, respectively.

Details on test conditions:

Test chambers were 250-mL glass beakers loosely covered with plastic Petri dish and filled with
approximately 220 mL of test water. The depth of the test water in a representative chamber was 7.5 cm.
All test chambers were labeled with the project number, test concentration and replicate designation. The
test chambers were impartially positioned by treatment group in a temperature-controlled environmental
chamber to maintain the target water temperature throughout the test period.
The test was conducted at a target water temperature of 20 ± 1C.
The test systems were illuminated using fluorescent tubes that emit wavelengths similar to natural
sunlight. The lights were controlled by an automatic timer to provide a photoperiod of 16 hours of light
and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights
went on and off to avoid sudden changes in light intensity.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

61 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

8.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

All daphnids in the negative control group appeared normal throughout the test.
Percent immobility in the 8.2, 13, 24, 47 and 108 mg a.i./L treatment groups at test termination was 0, 35,
15, 35 and 80%, respectively. Signs of toxicity observed among the surviving daphnids in the 8.2, 13, 24,
47 and 108 mg a.i./L treatment groups at test termination included floating and lethargy. Therefore in this
study, the no-immobility concentration was 8.2 mg a.i./L and the NOEC was <8.2 mg a.i./L. the 48-hour EC50 value was 61 mg a.i./L, with a 95% confidence interval
of 8.2 to 108 mg a.i./L.

Measured Concentrations ofFRET 13-0545in Test Solution Samples

 

Nominal Test Concentration (mg a.i./L)		Sample Number (549A-105-)	Sampling Time (Day)	Measured Concentration (mg a.i./L)^1,2	Percent of Nominal^2	Mean ± SD Measured Concentration (mg a.i./L)^3	Mean Measured Percent of Nominal^3
Negative Control		1	0 (New)	< LOQ	--	--	--
0		7	1 (Old)	< LOQ	--
		8	1 (Old)	< LOQ	--
		19	1 (New)	< LOQ	--
		25	2 (Old)	< LOQ	--
		26	2 (Old)	< LOQ	--
7.5		2	0 (New)	7.38	98.4	8.2 ± 1.01	109
		9	1 (Old)	7.51	100	(%CV =12.2)
		10	1 (Old)	7.16	95.5
			Average	7.34
		20	1 (New)	9.23	123
		27	2 (Old)	8.94	119
		28	2 (Old)	8.96	119
			Average	8.95
15		3	0 (New)	13.2	88.0	13 ± 0.411	87
		11	1 (Old)	12.9	86.0	(%CV = 3.24)
		12	1 (Old)	12.5	83.3
			Average	12.7
		21	1 (New)	12.2	81.3
		29	2 (Old)	12.4	82.7
		30	2 (Old)	12.7	84.7
			Average	12.6
30		4	0 (New)	23.9	79.7	24 ± 1.41	80
		13	1 (Old)	22.7	75.7	(%CV = 5.82)
		14	1 (Old)	22.5	75.0
			Average	22.6
		22	1 (New)	26.0	86.7
		31	2 (Old)	24.5	81.7
		32	2 (Old)	24.5	81.7
			Average	24.5

60		5	0 (New)	45.5	75.8	47 ± 1.11	79
		15	1 (Old)	44.4	74.0	(%CV = 2.36)
		16	1 (Old)	50.5	84.2
			Average	47.5
		23	1 (New)	47.7	79.5
		33	2 (Old)	49.1	81.8
		34	2 (Old)	46.7	77.8
			Average	47.9
120		6	0 (New)	117	97.5	108 ± 9.78	90
		17	1 (Old)	115	95.8	(%CV = 9.07)
		18	1 (Old)	115	95.8
			Average	115
		24	1 (New)	97.0	80.8
		35	2 (Old)	103	85.8
		36	2 (Old)	100	83.3
			Average	102
	^1  The method limit of quantitation (LOQ) of FRET 13-0545 in freshwater was set at 2.00 mg a.i./L, defined as the lowest analyte concentration in a sample at which the methodology has been validated and a mean recovery of 70-110% with a relative standard deviation of<20% has been obtained.  ^2Results were generated using Excel 2010. Manual calculations may differ slightly. ^3 This result was calculated as follows: [Day 0 (new) + Average Day 1 (old) + Day 1 (new) + Average of Day 2 (old)]/4

Cumulative Immobility and Observations

 

Mean Measured Concentration (mg a.i./L)	Rep.	No. Exposed	~5 Hours			24 Hours^3			48 Hours		Cumulative Percent Immobility
			Number Immobile^1	Observations^2		Number Immobile^1	Observations^2		Number Immobile^1	Observations^2
Negative	A	10	0	10 AN		0	10 AN		0	10 AN	0
Control	B	10	0	10 AN		0	10 AN		0	10 AN
8.2	A	10	0	4 Q,AN; 6 AN		0	4 Q,C; 6 AN		0	2 C; 4 Q,C; 4 AN	0
	B	10	0	2 Q,AN; 8 AN		0	10 AN		0	2 Q,C; 1 Q,AN; 7 AN
13	A	10	0	10 AN		0	10 AN		3	5 C; 1 Q,AN; 1 AN	35
	B	10	0	10 AN		0	10 AN		4	6 C
24	A	10	0	10 AN		0	5 C; 5 AN		2	8 C	15
	B	10	0	10 AN		0	7 C; 3 AN		1	9 C
47	A	10	0	5 Q,C; 5 AN		0	5 C; 5 Q,C		4	6 C	35
	B	10	0	6 Q,C; 4 Q,AN		0	4 C; 6 Q,C		2+1Q	5 C; 2 Q,C
108	A	10	0	7 C; 3 AN		1	8 C ; 1 Q,C		9	1 C	80
	B	10	0	5 C; 5 AN		1	9 C		7	3 C
^1  Cumulative number of immobile daphnids. Any immobile daphnids were removed from the test chambers at each observation interval. ^2  Observations of surviving organisms: AN = appear normal; C = lethargy; Q,AN = floating, trapped at water surface but appear normal after gentle submersion; Q,C = floating, trapped at water surface and appear lethargic after gentle submersion; Q = floating. ^3  Test solutions were renewed at approximately 24 hours.

EC₅₀ValuesBased on Mean Measured Test Concentrations

 

Time	EC50 (mg a.i./L)	95% Confidence Interval (mg a.i./L)	Statistical Method
24 Hours	> 108	--^1	NA^2
48 Hours	61	8.2 – 108	Binomial Analysis^3
^1 95% confidence limits could not be calculated with the immobility data obtained. ^2 NA = not applicable; <50% immobility precluded statistical calculation of an EC50value. ^3 The EC50value was estimated using non-linear interpolation between 47 and 108 mg a.i./L; the 95% confidence limits were determined by binomial probability.

Validity criteria fulfilled:

yes

Conclusions:

Following OECD TG 202, the cladoceran, Daphnia magna, was exposed for 48 hours under static-renewal conditions to five mean measured concentrations of FRET 13-0545 ranging from 8.2 to 108 mg a.i./L. Based on the mean measured concentrations, the 48-hour EC50 value was 61 mg a.i./L, with a 95% confidence interval of 8.2 to 108 mg a.i./L. The no-immobility concentration was 8.2 mg a.i./L and the NOEC was <8.2 mg a.i./L.

Executive summary:

According to OECD 202 and US EPA OCSPP 850.1010 test guidelines, Daphnia magna was exposed to test solution for 48 hr under static renewal conditions. Five exposrue concentrations were tested with nominal concentration of 7.5, 15, 30, 60 and 120 mg a.i./L.

Since the mean measured concentrations were outside the 80-120% range as recommended by guidelines, the results of the study were based on the mean measured concentrations.

Based on the mean measured concentrations, the 48-hour EC50 value was 61 mg a.i./L, with a 95% confidence interval of 8.2 to 108 mg a.i./L. The no-immobility concentration was 8.2 mg a.i./L and the NOEC was <8.2 mg a.i./L.

Description of key information

Following OECD TG 202, the cladoceran, Daphnia magna, was exposed for 48 hours under static-renewal conditions to five mean measured concentrations of FRET 13-0545 ranging from 8.2 to 108 mg a.i./L. Based on the mean measured concentrations, the 48-hour EC50 value was 61 mg a.i./L, with a 95% confidence interval of 8.2 to 108 mg a.i./L. The no-immobility concentration was 8.2 mg a.i./L and the NOEC was <8.2 mg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

61 mg/L