Heptanal, 2-(phenylmethylene)-, (2E)-

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February 2019 to 20 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines (OECD 211) and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

02 October 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 11, 33, 100, 300 and 900 µg/L & negative control
- Sampling method: Required volume of composite sample from all the test concentrations were collected on day of sampling and samples were sent to analytical team for analysis. In all cases, analyses were made on a sample from the same solution from all the groups, when freshly prepared and at renewal.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Serial dilution
- Eluate: Test Medium
- Differential loading: N/A
- Controls: Medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
- Other relevant information:
Test item stock of 6 mg/L was prepared by mixing 12 mg of test item in test medium and making up the final volume to 2000 mL using test medium.
Test medium (1 L) was used as negative control . To prepare test concentrations of 11, 33, 100, 300 and 900 µg/L, 1.834, 5.500, 16.667, 50 and 150 mL of test item stock was added and volume was made up to 1 L in a volumetric flask using test medium separately.
Based on the requirement, to prepare test concentrations of 11, 33, 100, 300 and 900 µg/L, 7.334, 22, 66.667, 200 and 600 mL of test item stock was added and volume was made up to 4 L in a volumetric flask using test medium separately. Test medium (4 L) was used as negative control.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Source: Ecotoxicology, Eurofins Advinus Limited, Bengaluru, India
- Maintenance: Daphnia were maintained in dilution medium (similar to one which was used in the test for acclimatization for at least three weeks as well as preparation of test medium). The test medium in which the Daphnia were maintained was changed at least twice in a week and they are fed with the alga, Pseudokirchneriella subcapitata.
- Neonates: At the start of the test, the animals were less than 24 hours old and were not first brood progeny. They were derived from a healthy stock (i.e., showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discoloured animals, etc.). The stock animals were maintained in culture conditions (light, temperature, medium, feeding and animals per unit volume) similar to those used in this test.
Adult gravid daphnids (which are already given their broods) were incubated individually in 100 mL of test medium, a day prior to start of the treatment. Progenies produced were observed (on the day of treatment) for neonate sex and after ascertaining the exclusive production of female progenies then the treatment was planned. Female daphnids aged less than 24 hours were used in the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

Guideline standard exposure duration

Hardness:

250 mg/L as CaCO3

Test temperature:

The test medium temperature was between 19.5 °C and 19.9 °C during the definitive test.

pH:

The pH was within the range 7.35 - 8.03 during the definitive test.

Nominal and measured concentrations:

Nominal Concentrations: 0.5, 5, 50, 100, 500 and 1000 µg/L
Measured Concentrations: 5, 11, 34, 97 and 331 µg/L (geometric mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: Beaksers
- Material, size, headspace, fill volume: 150 mL capacity, 100 mL test medium per replicate
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A - semi-static
- Renewal rate of test solution (frequency/flow rate): N/A
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test medium used in the test was Selenium medium with selenium and vitamin supplements (Selenium medium). The test medium was prepared using analytical or better grade reagents as per the following composition and procedure:
CaCl2.2H20: 293.8 g/L stolck - 1 mL/L of stock for medium preparation
MgSO4.7H20: 246.6 g/L stock - 0.5 mL/L of stock for medium preparation
NaHCO3: 64.8 g/L stock - 1 mL/L of stock for medium preparation
KCl: 58 g/L stock - 0.1 mL/L of stock for medium preparation
Na2SeO3: 0.0438 g/L stock - 0.05 mL/L of stock for medium preparation
Thiamine hydrochloride: 0.0750 g/L stock, Cyanocobalamine (B12): 0.010 g/L stock, Biotine: 0.0075 g/L stock (combined) - 0.1 mL/L of stock for medium preparation
Water (reverse osmosis water) was used for preparation of stock solutions and test medium. For preparation of stock solutions, each chemical was dissolved separately (except for Vitamin stock solution) in water and made up to 1 L. To prepare the Selenium medium, the stock solutions was mixed (as per the above composition) in approximately 800 mL water and made up the volume to 1 L. Test medium was aerated using aerators until the dissolved oxygen concentration equaled the air saturation value. The prepared test medium was used within 10 days from the date of preparation.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The dissolved oxygen concentration, temperature, pH and hardness were measured once a week in fresh and old media in the control, lowest and highest test item concentrations.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light: 8h dark
- Light intensity: 1307 - 1366 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality among the parent animals was recorded daily (± 2 h from treatment start on day 0 exposure) along with the behaviour (n: parent animal normal and b: parent animal abnormal). An animal was recorded as dead (mortality) when it is immobile, i.e. when it was not able to swim, or if there was no observed movement of appendages or post-abdomen within 15 seconds after gentle agitation of the test container.
The offspring produced by each parent animal were preferably removed and counted daily from the appearance of the first brood to prevent them consuming food intended for the parent. The numbers of living offspring were counted but the presence of aborted eggs and dead offspring were recorded. Offspring produced were observed for presence of male neonates (neonate sex) and ephippia.
The measurement of the length of the parent animals (i.e., body length excluding the anal spine) at the end of the test was measured. Time to production of first brood (and subsequent broods), number and size of broods per parent animal, number of aborted broods, and the intrinsic rate of population increase were also evaluated.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 0.5, 5, 50, 100, 500 and 1000 µg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

not specified

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

23.14 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % CI: 9.23 - 54.52 µg/L

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 11 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 34 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 34 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

There was no mortality of parent animals in the negative control. The mortality of parent animals was 0, 0, 0, 20 and 50 % on day 21 of exposure at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively.

The percent change in reproductive output of parent animals was -0.2, -2.7, -42.2, -60.6 and -99.2 % at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively. Reproductive output of parent animals evidenced statistically significant reduction in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.

Time to production of first brood (and subsequent broods), number and size of broods evidenced prolongation (production of broods) and reduction (number and size of broods) in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.

The percent production of male offspring was 0.5, 1.0, 1.2, 75.8, 78.3 and 100 % at the test concentrations of 0, 5, 11, 34, 97 and 331 µg/L, respectively. The data indicates that the production of male neonates was due to chemical induced stress in the test concentrations of 34, 97 and 331 µg/L. This was also evidenced by decline in the production of male neonates after third brood.

Measurement of the length of the parent animals at the end of the test (day 21) evidenced statistically significant reduction in the tested concentrations of 97 and 331 µg/L as compared with the control group.

Intrinsic rate of population increases evaluation evidenced reduction in the tested concentrations of 34 and 97 µg/L as compared with the control group. In the highest test concentration 331 µg/L, the intrinsic rate of population increases (r) was not evaluated due to absence of healthy offspring in this group (population not growing).

Male neonates and ephippia were recorded during the test. There were no instances of aborted eggs, aborted broods or dead offspring in the control and any of the test item treated groups.

Reported statistics and error estimates:

The estimation of EC50 values was not possible, since there was partial (20 and 50 %) mortality of parent animals observed at the tested concentrations.
The statistical analysis of the body length of the parent animals and juvenile production data was carried out using licensed copies of SYSTAT Statistical package version 12.0. The body length of the parent animals and offspring produced by each group was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Significant difference was tested using Dunnett’s test.
Data (total offspring in each group) was square root transformed prior to EC evaluation, since it’s a count data and follows Poisson distribution. The EC10, EC20, EC50 and EC90 value with 95% fiducial limits, and the equation for the dose-response curve was determined by Probit analysis by the method of Finney (1971) using in-house developed and validated computer programme.
Analysis of the number of living offspring per surviving parent animal, as per the OECD 211 Guideline, was not conducted as the data for total offspring in each group produced the most conservative results for hazard assessment.

 Definitive Test - Parent Animal Mortality

Group	Measured Conc. (µg/L)	No. of parent animal at start of test (1 per replicate)	No. of parent animal mortality on Day 21	% Mortality of parent animal on Day 21
G1	Negative control	10	0	0
G2	5	10	0	0
G3	11	10	0	0
G4	34	10	0	0
G5	97	10	2	20
G6	331	10	5	50

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L.

 Definitive Test - Offspring Production

Group	Measured Conc. (µg/L)	Offspring produced in a group										Total living offspring produced in a group	No. of live parent animals on day 21	No. of living offspring produced per live parent animal	No. of offspring produced in a group		% Reduction in offspring production
		PA1	PA2	PA3	PA4	PA5	PA6	PA7	PA8	PA9	PA 10				Mean	SD
G1	Negative control	78	84	80	82	87	86	82	86	88	89	842	10	84.2	84.2	3.6	-
G2	5	85	80	82	78	83	78	82	93	87	92	840	10	84.0	84.0	5.3	-0.2
G3	11	78	77	80	83	87	77	80	84	85	88	819	10	81.9	81.9	4.1	-2.7
G4	34	53	61	48	58	48	41	45	48	39	46	487*	10	48.7	48.7	6.9	-42.2
G5	97	40	41	18	29	33	15	49	35	34	38	332*	8	37.4	33.2	10.3	-60.6
G6	331	3	4	0	0	0	0	0	0	0	0	7$	5	1.4	0.7	1.5	-99.2

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L. There were no instances of aborted eggs, aborted broods or dead offspring in the control and any of the test item treated groups.

The coefficient of variation for control reproductive output is 4.3% (based on total number of living offspring per parent animal alive at the end of the test). *: Statistically significantly (p ≤ 0.05) lower than the respective control group.$:Statistical evaluation not undertaken due to less sample size.

Definitive Test - Broods Production

Group	Measured Conc. (µg/L)	Average No. of broods	Time to production of first brood and subsequent broods per parent animal				Time between brood production per parent animal			Size of broods per parent animal
			First	Second	Third	Fourth	1 - 2	2 - 3	3 - 4	First	Second	Third	Fourth
G1	Negative control	4	8.3	12.5	16.6	20.6	4.2	4.1	4	7.6	20.5	26.0	30.1
G2	5	4	8.5	12.5	16.6	20.6	4	4.1	4	8	19.9	25.4	30.7
G3	11	4	8.5	12.5	16.7	20.8	4	4.2	4.1	7.4	19.8	23.4	31.3
G4	34	2.8	12.1	16.5	20.6	No brood	4.4	4.4	No brood	21.8	15.1	14.8	No brood
G5	97	2.6	12.3	16.8	20.5	No brood	4.4	3.8	No brood	14.7	12.6	10.3	No brood
G6	331	0.4	17	21	No brood	No brood	4	No brood	No brood	1.5	2.0	No brood	No brood

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L.

 Definitive Test - Male Offspring Production and Intrinsic Rate of Population Increase

Group	Measured Conc. (µg/L)	Male offspring produced in a group										Total living offspring produced in a group	Total male offspring produced in a group	No. of male offspring produced in a group		% Male offspring produced in a group	Intrinsic Rate of Population Increase (r)
		PA1	PA2	PA3	PA4	PA5	PA6	PA7	PA8	PA9	PA10			Mean	SD
G1	Negative control	2	-	1	-	-	-	-	1	-	-	842	4	0.4	0.6	0.5	0.3255
G2	5	1	1	1	-	-	1	-	2	1	1	840	8	0.8	0.4	1.0	0.3241
G3	11	-	2	1	-	1	-	1	1	3	1	819	10	1.0	0.8	1.2	0.3184
G4	34	37	47	34	40	29	41	32	31	39	39	487	369	36.9	5.4	75.8	0.1191
G5	97	23	30	18	29	25	15	40	22	30	28	332	260	26.0	7.1	78.3	0.0935
G6	331	3	4	-	-	-	-	-	-	-	-	7	7	0.7	1.5	100	#

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L. #: Not evaluated due to absence of healthy offspring in this group.

 Definitive Test - Length of the Parent Animal

Replicate No.	Length of parent animal (mm)
	Group & Measured Concentration (µg/L)
	G1 & 0	G2& 5	G3& 11	G4& 34	G5 & 97	G6 & 331
1	4.29	4.34	4.31	4.16	3.61	2.67
2	4.36	4.36	4.23	4.20	3.79	2.74
3	4.42	4.37	4.28	4.27	-	2.11
4	4.34	4.26	4.37	4.16	3.89	-
5	4.38	4.39	4.39	4.07	3.91	-
6	4.41	4.36	4.19	4.21	-	2.01
7	4.44	4.31	4.28	4.25	3.81	-
8	4.35	4.38	4.36	4.08	3.72	-
9	4.36	4.33	4.39	4.18	3.95	2.13
10	4.31	4.38	4.33	4.19	3.84	-
Mean	4.37	4.35	4.31	4.18	3.82*	2.33*
SD	0.05	0.04	0.07	0.06	0.11	0.34

Note: Respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L.

*: Statistically significantly (p ≤ 0.05) lower than the respective control group.

 Test Item Content in Test Sample - Fresh Samples           

Date of receipt: 02 April 2019 (0 h sample) - Date of analysis: 02 April 2019

Dose (µg/L)	Sample	As a Test Item		% RSD	% Recovery	Mean ± SD	% RSD
		Test Item Conc. (µg/L)	Mean ± SD
11	G2R1-1	5.67530	5.44041 ± 0.352	6.5	51.59	49.46 ± 3.20	6.5
	G2R1-2	5.03552			45.78
	G2R1-3	5.61043			51.00
33	G3R1-1	5.96011	6.79191 ± 0.955	14	18.06	20.58 ± 2.90	14
	G3R1-2	6.58016			19.94
	G3R1-3	7.83546			23.74
100	G4R1-1	19.5194	22.7458 ± 2.83	12	19.52	22.75 ± 2.83	12
	G4R1-2	23.8937			23.89
	G4R1-3	24.8243			24.82
300	G5R1-1	58.8109	68.1128 ± 9.42	14	19.60	22.70 ± 3.14	14
	G5R1-2	67.8758			22.63
	G5R1-3	77.6517			25.88
900	G6R1-1	399.1496	483.763 ± 79.2	16	44.35	53.75 ± 8.80	16
	G6R1-2	496.0824			55.12
	G6R1-3	556.0570			61.78

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Aged Samples

Date of receipt: 03 April 2019 (24 h sample) - Date of analysis: 03 April 2019

Dose (µg/L)	Sample	As a Test Item		% RSD	% Recovery	Mean ± SD	% RSD
		Test Item Conc. (µg/L)	Mean ± SD
11	G2R1-1	4.44827	5.71351 ± 1.11	19	40.44	51.94 ± 10.1	19
	G2R1-2	6.16374			56.03
	G2R1-3	6.52851			59.35
33	G3R1-1	3.59673	5.51061 ± 2.04	37	10.90	16.70 ± 6.19	37
	G3R1-2	7.66347			23.22
	G3R1-3	5.27162			15.97
100	G4R1-1	9.0020	9.9615 ± 0.912	9.2	9.002	9.96 ± 0.912	9.2
	G4R1-2	10.0661			10.07
	G4R1-3	10.8163			10.82
300	G5R1-1	47.7794	41.8375 ± 7.71	18	15.93	13.95 ± 2.57	18
	G5R1-2	33.1316			11.04
	G5R1-3	44.6016			14.87
900	G6R1-1	99.2060	119.689 ± 23.2	19	11.02	13.30 ± 2.58	19
	G6R1-2	144.916			16.10
	G6R1-3	114.943			12.77

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

 

Test Item Content in Test Sample - Fresh Samples           

Date of receipt: 04 April 2019 (0 h sample) - Date of analysis: 04 April 2019

Dose (µg/L)	Sample	As a Test Item		% RSD	% Recovery	Mean ± SD	% RSD
		Test Item Conc. (µg/L)	Mean ± SD
11	G2R1-1	3.01164	3.21714 ± 0.223	6.9	27.38	29.25 ± 2.03	6.9
	G2R1-2	3.18527			28.96
	G2R1-3	3.45450			31.40
33	G3R1-1	8.19345	7.75416 ± 0.745	10	24.83	23.50 ± 2.26	10
	G3R1-2	6.89394			20.89
	G3R1-3	8.17508			24.77
100	G4R1-1	22.8310	21.5255 ± 2.48	12	22.83	21.53 ± 2.48	12
	G4R1-2	23.0777			23.08
	G4R1-3	18.6678			18.67
300	G5R1-1	56.6449	57.8544 ± 3.76	6.5	18.88	19.28 ± 1.25	6.5
	G5R1-2	62.0682			20.69
	G5R1-3	54.8500			18.28
900	G6R1-1	189.339	219.205 ± 26.5	12	21.04	24.36 ± 2.95	12
	G6R1-2	239.993			26.67
	G6R1-3	228.284			25.36

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Aged Samples

Date of receipt: 05 April 2019 (24 h sample) - Date of analysis: 05 April 2019

Dose (µg/L)	Sample	As a Test Item		% RSD	% Recovery	Mean ± SD	% RSD
		Test Item Conc. (µg/L)	Mean ± SD
11	G2R1-1	0.562052	0.569688 ± 0.0334	5.9	5.110	5.179 ± 0.304	5.9
	G2R1-2	0.540781			4.916
	G2R1-3	0.606231			5.511
33	G3R1-1	0.353196	0.408089 ± 0.0480	12	1.070	1.237 ± 0.150	12
	G3R1-2	0.429035			1.300
	G3R1-3	0.442036			1.340
100	G4R1-1	1.99137	1.72117 ± 0.238	14	1.991	1.721 ± 0.238	14
	G4R1-2	1.54459			1.545
	G4R1-3	1.62756			1.628
300	G5R1-1	3.74099	3.86265 ± 0.647	17	1.247	1.288 ± 0.220	17
	G5R1-2	4.56218			1.521
	G5R1-3	3.28477			1.095
900	G6R1-1	61.67154	53.82243 ± 8.901	17	6.852	5.980 ± 0.989	17
	G6R1-2	44.15122			4.906
	G6R1-3	55.64455			6.183

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Fresh Samples           

Date of receipt: 09 April 2019 (0 h sample) - Date of analysis: 09 April 2019

Dose (µg/L)	Sample	As a Test Item		% RSD	% Recovery	Mean ± SD	% RSD
		Test Item Conc. (µg/L)	Mean ± SD
11	G2R1-1	9.84758	9.83686 ± 0.457	4.6	89.52	89.43 ± 4.16	4.6
	G2R1-2	9.37445			85.22
	G2R1-3	10.2885			93.53
33	G3R1-1	30.6872	29.3491 ± 1.17	4.0	92.99	88.94 ± 3.54	4.0
	G3R1-2	28.5317			86.46
	G3R1-3	28.8284			87.36
100	G4R1-1	98.7444	92.5732 ± 6.90	7.5	98.74	92.57 ± 6.90	7.5
	G4R1-2	93.8492			93.85
	G4R1-3	85.1258			85.13
300	G5R1-1	222.996	251.574 ± 25.1	10	74.33	83.86 ± 8.37	10
	G5R1-2	270.054			90.02
	G5R1-3	261.672			87.22
900	G6R1-1	803.163	840.155 ± 32.5	3.9	89.24	93.35 ± 3.61	3.9
	G6R1-2	853.463			94.83
	G6R1-3	863.841			95.98

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Aged Samples           

Date of receipt: 10 April 2019 (24 h sample) - Date of analysis: 10 April 2019

Dose (µg/L)	Sample	As a Test Item		% RSD	% Recovery	Mean ± SD	% RSD
		Test Item Conc. (µg/L)	Mean ± SD
11	G2R1-1	4.66025	4.28493 ± 0.550	13	42.37	38.95 ± 5.00	13
	G2R1-2	3.65314			33.21
	G2R1-3	4.54139			41.29
33	G3R1-1	12.4176	14.0324 ± 2.10	15	37.63	42.52 ± 6.36	15
	G3R1-2	13.2742			40.22
	G3R1-3	16.4053			49.71
100	G4R1-1	41.8545	40.9656 ± 1.01	2.5	41.85	40.97 ± 1.01	2.5
	G4R1-2	41.1685			41.17
	G4R1-3	39.8739			39.87
300	G5R1-1	102.035	112.839 ± 10.7	9.5	34.01	37.61 ± 3.57	9.5
	G5R1-2	113.013			37.67
	G5R1-3	123.470			41.16
900	G6R1-1	309.525	370.765 ± 62.2	17	34.39	41.20 ± 6.91	17
	G6R1-2	368.839			40.98
	G6R1-3	433.931			48.21

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion: There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Fresh Samples           

Date of receipt: 16 April 2019 (0 h sample) - Date of analysis: 17 April 2019

Dose (µg/L)	Sample	As a Test Item		% RSD	% Recovery	Mean ± SD	% RSD
		Test Item Conc. (µg/L)	Mean ± SD
11	G2R1-1	10.3359	9.69719 ± 0.685	7.1	93.96	88.16 ± 6.23	7.1
	G2R1-2	8.97405			81.58
	G2R1-3	9.78162			88.92
33	G3R1-1	30.6844	32.1495 ± 3.98	12	92.98	97.42 ± 12.1	12
	G3R1-2	29.1063			88.20
	G3R1-3	36.6577			111.1
100	G4R1-1	86.6861	83.9817 ± 3.61	4.3	86.69	83.98 ± 3.61	4.3
	G4R1-2	85.3718			85.37
	G4R1-3	79.8873			79.89
300	G5R1-1	263.052	268.811 ± 16.0	6.0	87.68	89.60 ± 5.34	6.0
	G5R1-2	256.465			85.49
	G5R1-3	286.916			95.64
900	G6R1-1	775.845	760.511 ± 31.0	4.1	86.21	84.50 ± 3.45	4.1
	G6R1-2	724.793			80.53
	G6R1-3	780.893			86.77

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Test Item Content in Test Sample - Aged Samples           

Date of receipt: 17 April 2019 (24 h sample) - Date of analysis: 17 April 2019

Dose (µg/L)	Sample	As a Test Item		% RSD	% Recovery	Mean ± SD	% RSD
		Test Item Conc. (µg/L)	Mean ± SD
11	G2R1-1	2.43400	2.80298 ± 0.354	13	22.13	25.48 ± 3.22	13
	G2R1-2	2.83417			25.77
	G2R1-3	3.14076			28.55
33	G3R1-1	9.90250	9.80214 ± 0.409	4.2	30.01	29.70 ± 1.24	4.2
	G3R1-2	10.15169			30.76
	G3R1-3	9.35223			28.34
100	G4R1-1	32.5757	32.4508 ± 4.99	15	32.58	32.45 ± 4.99	15
	G4R1-2	27.4003			27.40
	G4R1-3	37.3763			37.38
300	G5R1-1	81.8398	86.8849 ± 6.93	8.0	27.28	28.96 ± 2.31	8.0
	G5R1-2	84.0283			28.01
	G5R1-3	94.7866			31.60
900	G6R1-1	252.120	243.533 ± 15.6	6.4	28.01	27.06 ± 1.73	6.4
	G6R1-2	225.542			25.06
	G6R1-3	252.936			28.10

Acceptance criteria:The criteria for acceptance of analysis results of the test concentration should be 80 to 120% of the claimed concentration with ≤ 20 % RSD of analyzed concentration.

Conclusion:There was no interference in the G1 group samples at the retention time of the analyte. The obtained results of all the groups were out of acceptance limits (80-120%).

Validity criteria fulfilled:

yes

Conclusions:

The reproduction EC10 of Amyl Cinnamic Aldehyde on Daphnia magna is 23.14 µg/L.
The reproduction NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 11 µg/L.
The reproduction LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 34 µg/L.
The lethal effects (mortality of parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 34 µg/L.
The lethal effects (mortality of parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.
The growth effects (length of the parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 34 µg/L.
The growth effects (length of the parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.

Executive summary:

The reproductive effects of the test item, Amyl Cinnamic Aldehyde were studied on the reproductive output of daphnids, Daphnia magna. This test was conducted as a semi-static test with renewal of test medium at every 24 h interval. Mortality, behaviour and offspring produced (neonate sex) of the parent animals were observed daily during the 21 day test.

A range finding test was carried out to determine the test concentrations for the definitive test. Female, Daphnia magna aged less than 24 hours old were exposed to the nominal test item concentrations of 0.5, 5, 50, 100, 500 and 1000 µg/L along with a negative control in test medium (selenium medium). There was no mortality of parent animals in the negative control and any of the test item treated groups. The percent change in reproductive output of parent animals was 1, 3, 1, -41, -52 and -96 % at the tested concentrations of 0.5, 5, 50, 100, 500 and 1000 µg/L, respectively.

In definitive test, Daphnia magna (female) less than 24 hours old were exposed to nominal test item concentrations of 11, 33, 100, 300 and 900 µg/L along with a negative control in test medium.

The stability test results concluded that the test item was not stable in the selenium medium after 24 h in the claimed concentrations of 0.5167 and

1035.4 µg/L.

The results of active ingredient analysis of test concentrations of 11, 33, 100, 300 and 900 µg/L showed that overall percent agreement was as below:

Sample	Mean Minimum (%)	Mean Maximum (%)
0 h Fresh	14.46	101.9
24 h Aged	0.9701	51.95

Since the deviation from the measured initial concentration was greater than ± 20%. The results were expressed in terms of the geometric mean concentration. Geometric mean test concentrations are 5, 11, 34, 97 and 331 µg/L (respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L).

There was no mortality of parent animals in the negative control. The mortality of parent animals was 0, 0, 0, 20 and 50 % on day 21 of exposure at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively.

The percent change in reproductive output of parent animals was -0.2, -2.7, -42.2, -60.6 and -99.2 % at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively. Reproductive output of parent animals evidenced statistically significant reduction in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.

Time to production of first brood (and subsequent broods), number and size of broods evidenced prolongation (production of broods) and reduction (number and size of broods) in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.

The percent production of male offspring was 0.5, 1.0, 1.2, 75.8, 78.3 and 100 % at the test concentrations of 0, 5, 11, 34, 97 and 331 µg/L, respectively. The data indicates that the production of male neonates was due to chemical induced stress in the test concentrations of 34, 97 and 331 µg/L. This was also evidenced by decline in the production of male neonates after third brood.

Measurement of the length of the parent animals at the end of the test (day 21) evidenced statistically significant reduction in the tested concentrations of 97 and 331 µg/L as compared with the control group.

Intrinsic rate of population increases evaluation evidenced reduction in the tested concentrations of 34 and 97 µg/L as compared with the control group. In the highest test concentration 331 µg/L, the intrinsic rate of population increases (r) was not evaluated due to absence of healthy offspring in this group (population not growing).

Male neonates and ephippia were recorded during the test. There were no instances of aborted eggs, aborted broods or dead offspring in the control and any of the test item treated groups.

The effects of Amyl Cinnamic Aldehyde on Daphnia magna reproduction are as below:

Reproduction Endpoints	Test Item (µg/L)	95% Fiducial Limits (µg/L)
		Lower	Upper
NOEC	> 11	-	-
LOEC	34	-	-
EC10	23.14	9.23	54.52
EC20	38.25	15.26	90.13
EC50	100.04	39.91	235.74
EC90	432.54	172.56	1019.23
Regression Equation (Y = A + BX)		Y = 0.97 + 2.02 X

The reproduction EC10 of Amyl Cinnamic Aldehyde on Daphnia magna is 23.14 µg/L.

The reproduction NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 11 µg/L.

The reproduction LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 34 µg/L.

The lethal effects (mortality of parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 34 µg/L.

The lethal effects (mortality of parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.

The growth effects (length of the parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is above 34 µg/L.

The growth effects (length of the parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.

Description of key information

EC10 (reproduction) = 23.14 µg/L; OECD 211; Halappa, 2019

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

23.14 µg/L

Additional information

OECD 211 (Halappa, 2019)

The reproductive effects of the test item to Daphnia magna were assessed according to OECD Guideline 211 (2012).

Daphnia magna (female) less than 24 hours old were exposed to nominal test item concentrations of 11, 33, 100, 300 and 900 µg/L along with a negative control in test medium.  The stability test results concluded that the test item was not stable in the selenium medium and a semi-static exposure scenario was used (24 h renewal). Since the deviation from the measured initial concentration was greater than ± 20%, results were expressed in terms of the geometric mean concentration. Geometric mean test concentrations are 5, 11, 34, 97 and 331 µg/L (respective nominal test concentrations are 11, 33, 100, 300 and 900 µg/L).

There was no mortality of parent animals in the negative control. The mortality of parent animals was 0, 0, 0, 20 and 50 % on day 21 of exposure at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively.  The percent change in reproductive output of parent animals was -0.2, -2.7, -42.2, -60.6 and -99.2 % at the test concentrations of 5, 11, 34, 97 and 331 µg/L, respectively.  Reproductive output of parent animals evidenced statistically significant reduction in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.  Time to production of first brood (and subsequent broods), number and size of broods evidenced prolongation (production of broods) and reduction (number and size of broods) in the tested concentrations of 34, 97 and 331 µg/L as compared with the control group.  The percent production of male offspring was 0.5, 1.0, 1.2, 75.8, 78.3 and 100 % at the test concentrations of 0, 5, 11, 34, 97 and 331 µg/L, respectively. The data indicates that the production of male neonates was due to chemical induced stress in the test concentrations of 34, 97 and 331 µg/L. This was also evidenced by decline in the production of male neonates after third brood.  Measurement of the length of the parent animals at the end of the test (day 21) evidenced statistically significant reduction in the tested concentrations of 97 and 331 µg/L as compared with the control group.  Intrinsic rate of population increases evaluation evidenced reduction in the tested concentrations of 34 and 97 µg/L as compared with the control group. In the highest test concentration 331 µg/L, the intrinsic rate of population increases (r) was not evaluated due to absence of healthy offspring in this group (population not growing).  Male neonates and ephippia were recorded during the test. There were no instances of aborted eggs, aborted broods or dead offspring in the control and any of the test item treated groups.

The reproduction EC10 of Amyl Cinnamic Aldehyde on Daphnia magna is 23.14 µg/L.

The reproduction NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is > 11 µg/L.

The reproduction LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 34 µg/L.

The lethal effects (mortality of parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is > 34 µg/L.

The lethal effects (mortality of parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.

The growth effects (length of the parent animals) NOEC of Amyl Cinnamic Aldehyde on Daphnia magna is > 34 µg/L.

The growth effects (length of the parent animals) LOEC of Amyl Cinnamic Aldehyde on Daphnia magna is 97 µg/L.