LUMIPOD

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:

supporting study

Study period:

From October 17, 2022 to November 14, 2022

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The lowest and highest test item loading rates be analysed when freshly prepared and at the time of renewal on one occasion during the first week of the test (i.e. analyses should be made on a sample from the same solution - when freshly prepared and at renewal). These determinations were repeated at least at weekly intervals thereafter.

Details on test solutions:

Test solutions of required concentrations 3.2 and 10.2 mg/L were prepared by direct addition (by weighing) from the test item with the M4 medium. In order to facilitate the weight (viscous product), the test item was deposited on a glass slide and then, introduced into the glass flask for the mixing protocol.
The prepared samples were stirred 4 hours at 20 ± 2°C and at darkness. After a settling period of 15 minutes, the samples were sampled at the middle of the glass flask. The solutions obtained were named WAF (Water Accommodated Fraction).

It was not possible to prepare each WAF individually due to the limitations of weighing small amounts of UVCB and/or the large test solution volumes required.
According to Guidance document on aquatic toxicity testing of difficult substances and mixtures OECD n°23 [8], serial dilution of a single stock WAF may be the only practical approach; however, these reasons for this departure from standard practice were fully described in this test report.
Therefore, the concentrations 0.1 mg/L - 0.3 mg/L – 1.0 mg/L were prepared by dilution of the stock WAF solutions 3.2 or 10.2 mg/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna Straus, clone number 5 at least the third generation (i.e. the third brood) obtained with a non-cyclic parthenogenesis reproduction.
- Age at study initiation (mean and range, SD): daphnids less than 24 hours old
- Length at study initiation (length definition, mean, range and SD): animals from a size between 560 µm and 800 µm
- Method of breeding: The daphnids breeding was carried out in closed bottles placed in a climatic chamber free from any toxic vapour and in medium M4.
- Feeding during test: It was done at least 3 times per week (i.e. corresponding to media changes). A ration of mixture of 2 algal cells (Chlorella vulgaris and Pseudokirchneriella subcapitata) between 0.1 and 0.2 mg
Carbon/Daphnia/Day is recommended. The ration was determined to obtain 60 offspring per parent animal in the controls.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

21 d

Remarks on exposure duration:

The test was in semi-static conditions, all the medium were renewed every 48 hours.
The test duration was 21 days.

Hardness:

250-260 mg/L
Hardness was measured at least once a week, in fresh and old media, in the Control, the lowest and the highest test substance concentration.

Test temperature:

18-22°C (range ± 1°C)
An additional test vessel was used for the purpose of temperature monitoring.

pH:

7.7-8.8
pH values were measured at least once a week, in fresh and old media, in the Control, the lowest and the highest test substance concentration.

Nominal and measured concentrations:

Five concentrations: 0.1 mg/L - 0.3 mg/L – 1.0 mg/L – 3.2 mg/L and 10.2 mg/L nominal test item loading rate /L, plus a Control (Test medium only) for the definitive test.

Details on test conditions:

Protocole
Protocol consisted of preparing a range of five test item concentrations in test medium M4 and a control, all in 10 replicates in glass flasks: one animal, young female daphnia aged less than 24 hours at the start of the test per flask (1 daphnia / flask).
The test was in semi-static conditions, all the medium were renewed every 48 hours.
The test duration was 21 days.
Test medium: M4 ELENDT

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Conc. based on:

test mat.

Dose descriptor:

LOELR

Effect conc.:

0.3 mg/L

Conc. based on:

test mat.

Dose descriptor:

EL50

Effect conc.:

2.2 mg/L

Conc. based on:

test mat.

Details on results:

The measured concentrations of the test item varied by more than ± 20 % of nominal or initial measured concentration during the test.

Chemical analysis in daphnids solutions

The table below summarizes the results achieved and the variation between initial measurement and before renewal.

		Control (mg/L)	0.10 mg/L (nominal test item loading rate)	10.2 mg/L (nominal test item loading rate)
First week October 17 to 19, 2022	At initial (Fresh)	<0.06	0.084	5.24
First week October 17 to 19, 2022	Before renewal (Old)	<0.06	<0.06	1.01
Variation (%)	-	-	>28.6	-80.7
Second week October 25 to 27, 2022	At initial (Fresh)	<0.06	0.073	6.70
Second week October 25 to 27, 2022	Before renewal (Old)	<0.06	<0.06	<0.63
Variation (%)	-	-	>17.8	> 90.6
Third week November 06 to 07, 2022	At initial (Fresh)	<0.06	0.063	7.11
Third week November 06 to 07, 2022	Before renewal (Old)	<0.06	<0.06	0.63
Variation (%)	-	-	NA	-91.1

At the nominal concentration of 0.10 mg/L, the test item  was not  considered as stable in the testing conditions because the measured concentrations of the test item varied more than ± 20 % of initial measured concentration during the test.

At the nominal concentration of 10.2 mg/L, the test item  was not  considered as stable in the testing conditions because the measured concentrations of the test item varied more than ± 20 % of initial measured concentration during the test.

Validity criteria fulfilled:

yes

Conclusions:

The coefficient of variation around the mean number of living offspring produced by parent animal in the controls was calculated. It is ≤ 25%, if the test was well run:

Coefficient of variation for control fecundity 13.1%

The results obtained show that test item “L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride” has effect on the reproductive output of daphnids under the studied conditions and give an EL50 = 2.2 mg test item / L [1.6 - 3.5].

Results (in mg nominal test item loading rate) / L of test medium)
NOELR 0.1 mg/L
LOELR 0.3 mg/L
EL50 -21 days [Confidence interval 95%] 2.2 mg/L [ 1.6 – 3.5 ]

Validity criteria for OECD 211 were satisfied.

Executive summary:

The aim of this study was to assess the effects of the test item “L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride” on the reproductive output of Daphnia magna, according to the OECD 211 guideline.

 

This study was carried out in accordance with the study plan no.21/1157F/d dated October 13, 2022.

There were three deviations (see § 9.3) and no amendment during this study.

 

At the end of the test, the total number of living offspring produced per animal alive at the end of the test was assessed.

The reproductive output of the animals exposed to the test item was compared to the control in order to determine the Lowest Observed Effect Loading rate (LOELR) and hence the No Observed Effect Loading rate (NOELR). In addition and as far as possible, the data were analysed using a regression model in order to estimate the concentration that would cause a X% reduction in reproductive output, i.e. ECx.

 

In agreement with the sponsor and according to the results of the study 21/1157F/c, the chemical analysis of “L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride” in the test solutions was performed by GC/FID in order to check the test item stability throughout the study.

 

Results are presented as follows:

Number of living offsprings at 21 days in each flask
Nominal test item loading rates	Control	0.1 mg/L	0.3 mg/L	1.0 mg/L	3.2 mg/L	10.2 mg/L
Total of living offsprings	86	73	49	25	33	17
	77	62	0	35	30	(43)
	80	55	43	46	38	34
	70	62	78	56	52	43
	71	63	43	30	30	46
	73	63	73	58	28	(48)
	102	60	43	53	28	47
	72	83	52	32	54	38
	71	57	53	30	31	45
	70	58	64	45	53	34
Mean of living offsprings	77.2	63.6	49.8	41	37.7	38.0
Total Aborted offsprings	0	0	0	19	45	51
Dead parent animal	/	/	/	/	/	2

 

The results obtained show that test item “L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride” has effect on the reproductive output of daphnids under the studied conditions and give an EL50 = 2.2 mg test item / L [1.6 - 3.5].

 

Results (in mg nominal test item loading rate) / L of test medium)

NOELR	0.1 mg/L
LOELR	0.3 mg/L
EL 50 - 21 days  [Confidence interval 95%]	2.2 mg/L [ 1.6 – 3.5 ]

 

Validity criteria conformity:

Criteria	Validity for OECD 211	Validity for the test
Mortality of parent animals in the controls (female Daphnia) at the end of the test	≤ 20%	Ok (0%)
Mean number of live offspring produced per animal surviving in the controls	≥ 60	Ok (77.2)

 

All the validity criteria were successful.

Description of key information

The results obtained show that test item “L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride” has effect on the reproductive output of daphnids under the studied conditions and give an EL50 = 2.2 mg test item / L [1.6 - 3.5].

Due to the instability of the registered substance during the test system, we consider that  the NOELR = 0.1 mg/L define the hazard of the degradation products and will not be used for the classification of the parent substance. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.1 mg/L