Reaction mass of (3aR,5aS,9aS,9bR)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan and (3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 April 2021 to 25 October 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

(incl. QA statement) inspected on 12 - 15 November 2019 / signed on 27 August 2020

Specific details on test material used for the study:

Dry area, protected from light, in refrigerator (2 - 8 °C), store under
nitrogen in a closed container after first opening

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

The saturated solution and the control were analyzed via GC-MS at 5 times during the test (at least once within a period of 7 days) in the fresh media at the start of an renewal interval (0 hours) as well as in the old media at the end of an renewal interval (24 hours).
The samples were analyzed under GLP with a GC-MS method which has been implemented under non-GLP and documented finally in the GLP raw data. The GC-MS method was validated according to Annex I. For the analytical methods and the results of the method validation see section 14 and Annex I.
At the start of the renewal intervals, samples of the fresh media monitoring were taken after preparation of the saturated solution and analyzed. At the end of the renewal intervals (24 hours), samples of the old media were taken from the test vessels.

Vehicle:

no

Details on test solutions:

A saturated solution with a nominal concentration of 10 mg/L was prepared 24 ± 1 hours before the start of the exposure and each renewal day.
For the saturated solution an appropriate amount of the test item was weighed out and transferred into a glass bottle with an appropriate amount of dilution water (see Table 2). The dispersion was shaken for 24 ± 1 hours (20 rpm, room temperature). The dispersion was removed by siphoning from the middle of the water column and were centrifuged (17000 g for 20 minutes at 20 ± 1 °C) to separate undissolved test item.
The saturated solution was checked via laser beam (Tyndall effect) . No undissolved test item was observed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Age at study initiation (mean and range, SD):
Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.
- Method of breeding:
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx
Culture medium: Elendt M4, according to OECD 211,
- Source: Daphnia magna STRAUS, obtained from continuous laboratory cultures. Origin =
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany.
- Culture feeding: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus,. The algae are cultured at the test facility.
- Feeding during test: Yes. Daily feeding with unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, 0.2 mg C / daphnia and day

ACCLIMATION
Acclimatization was not necessary, because the composition of the dilution water is equivalent to the culture medium

Test type:

semi-static

Water media type:

freshwater

Remarks:

edium Elendt M4 according to OECD 211

Limit test:

yes

Total exposure duration:

21 d

Hardness:

The total hardness was above 140 mg/L as CaCO3.

Test temperature:

Measured temperature between 20.1 and 20.8°C. The temperature was within the range of 20 ± 2 °C and not vary by more than 2°C.

pH:

The pH in the control was between 7,43 and 8.11
The pH in the tested concentration was between 7.49 and 7.92.
Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.

Dissolved oxygen:

The dissolved oxygen in the control was between 8.67 and 8.90 at test start and between 7.33 et 7.88 at test end
The pH in the tested cocnentration was between 8.22 and 8.80 at test start and between 7.00 and 8.25 at test end. The dissolved oxygen concentration was above 3 mg/L.

Salinity:

not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

nominal concentration : Saturated solution at 10 mg/L
Time weighted mean measured test itemconcentration : saturated solution 0.937 mg/l

Details on test conditions:

TEST SYSTEM :
Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw were used and filled up to the top with the test solutions. A test volume of approximately 130 mL was provided in each test vessel.
Test water : Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 211
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST CONDITION :
Test temperature (target) The temperature should be in the range of 18 – 22°C and should not vary by more than 2 °C.
Light intensity (target) Max. 1500 lx
Photoperiod 16/8 hours light/dark cycle
Aeration Test vessels were not aerated during the test.
Feeding Daily feeding with unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, max. 0.2 mg C / daphnia and day

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality of parental daphnids: The number of parental daphnids, which were immobilized or dying during the test and the dates of death were observed and recorded once a day at least at the same time as the offspring was counted. Dead daphnids were removed as soon as they were detected. Accidental mortality did not occur.

- Offspring Trials were checked daily for first appearance of juveniles. The number of neonates (alive and dead progeny) was counted and the dates of release were recorded once a day from the first day of production of neonates until the end of the exposure. The neonates (alive and dead) were removed after counting and before addition of algae to prevent them from consuming food intended for the adults. The number of aborted eggs or dead offspring and the dates of
observation were recorded. At the end of the test (21 days), the total number of living offspring was assessed.
- Additional observations / condition of parental daphnids: Abnormalities (e.g. swimming behavior, number of males and winter eggs) were observed and recorded on each day of observation.
- Total body length / Mean dry weight of parental daphnids: At the end of the test, the total body length excluding the anal spine of each survived parental daphnid and the mean dry weight of the
survived parental daphnids of all loading rates and the control were determined. As stated in the OECD protocol, this may be useful additional information but is not used for determination of a NOEC.
- Additional endpoints: The time to first brood, the intrinsic rate of population increase and the number and size of first brood per animal were reported, but not used for endpoint calculations.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.937 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.937 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.937 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.937 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

The number of juveniles in all replicates of the control and the test group was counted and recorded every day.
The average number of living juveniles at the end of the test after 21 days per survived parental female daphnid was 159 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.
Accidental and inadvertent mortality as defined in the OECD test guideline were not observed.
The effects on reproduction were evaluated based on the reproduction per female parent animal inserted at the start of the exposure. The effect values for the cumulative offspring per introduced parent are reported.
No statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the saturated solution (0.937 mg/L). Therefore, the LOEC was determined to be > 0.937 mg/L and the NOEC ≥ 0.937 mg/L for the cumulative offspring per introduced parent.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

The NOEC and LOEC for the reproduction were determined with the software ToxRat Professional as specified below. Significant differences of reproduction were determined in comparison to the
control using statistical standard procedures as normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test) and a two sample t-test for reproduction. P-values of the normality and the variance homogeneity test were 0.01.
For the effects on reproduction, the juveniles per introduced parent at the end of the test excluding accidental and inadvertent mortality (reproductive output) were counted. Since no accidental or inadvertent mortality was observed, no parental daphnids were excluded from the evaluation of the reproductive output.
The coefficients of variation around the mean number of living offspring produced per introduced parent and per survived parent in the control and the test groups were evaluated. The NOEC and LOEC for the adult mortality were empirically derived from the observed adult mortality data.

Validity criteria :

Mortality of the parent animal in the control group should not exceed 20% at the end of the test. No parent animal in the control group died. Actual mortality of the parent animals in the control group was 0%.
The mean number of living offspring produced per survived parent animal at the end of the test was 159 in the control group (required: at least 60 juveniles per survived parental daphnid).

Water quality : 

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits.

 pH-Values

 

Test group	Fresh	Old	Fresh	Old	Fresh	Old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
0.937 mg/l	7.51	7.91	7.49	7.91	7.63	7.92
Control	7.47	7.99	7.45	7.95	7.57	7.99

The pH-values should be in the range of 6 – 9. Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.

Dissolved Oxygen Concentration [mg/L]

 

Test group	Fresh	Old	Fresh	Old	Fresh	Old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
0.937 mg/l	8.62	8.17	8.80	8.25	8.22	7.00
Control	8.67	7.37	8.90	7.88	8.79	7.33

 The dissolved oxygen concentration was above 3 mg/L.

Total Hardness [mg/L] as CaCO3

Test group	Fresh	Old	Fresh	Old	Fresh	Old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
0.937 mg/l	286	253	232	231	247	248
Control	267	252	241	234	253	251

 The total hardness was above 140 mg/L as CaCO3.

Temperature [°C]

Test group	Fresh	Old	Fresh	Old	Fresh	Old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
0.937	20.8	20.8	20.4	20.7	20.3	20.5
Control	20.8	20.7	20.1	20.6	20.5	20.6

 The temperature was within the range of 20 ± 2 °C.

 

Effects on Adult mortality and Reproduction for Surviving Parents

(based on the nominal loading rates of the test item)

 

Nominal loading rate of the test item (mg/L)	Mean number of offspring per survived parental daphnid
	Mean	SD	CV
0.937	146	9.46	6.5
control	159	21.5	13.6

 

 

Mortality (%) of the Adult Daphnids after 7, 14 and 21 days of exposure

(control: n = 10; test item loading rates: n = 10)

 

Nominal loading rate of the test item (mg/L)	Adult mortality (%)
	7 days	14 days	21 days
0.937	0	0	0
Control	0	0	0

 

Intrinsic rates of natural increase

 

Nominal loading rate of the test item (mg/L)	Mean IR MV±SD	CV (%)
0.937 Control	0.399±0.0177 0.434±0.0330	4.4 7.6

 

First appearance of living juveniles at the Survived Parental Daphnids in the Individual Groups

 

Nominal loading rate of the test item (mg/L)	Day of first appearance of living juveniles at the survived parental daphnids in replicate no.										First appearance mean day
	1	2	3	4	5	6	7	8	9	10
0.937 Control	8 8	8 8	8 8	8 8	8 8	8 7	8 8	8 7	8 9	8 8	8.0 7.9

 

 

 

 Total Body Length and Dry Weight of the Survived Parental Daphnids

 

Nominal loading rate of the test item (mg/L)	Total length of the survived parent animals (mm) Replicate no.										MV (mm)	N	Dry weight (mg)
	1	2	3	4	5	6	7	8	9	10			Ʃ	MV
0.937 Control	4.5 5.0	4.5 5.0	4.5 5.0	4.5 5.0	4.5 5.0	5.0 4.5	5.0 5.0	4.5 4.5	5.0 4.5	4.5 5.0	4.65 4.85	10 10	10.5 11.8	1.05 1.18

N = number of the survived parental female daphnids

 

Analytical results : 

The limit test item concentration and the control were analytically verified via GC-MS five times
during the test in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7, 8, 14 and 15) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 9, 15 and 16).

Sampling date	Day 0 Fresh medium 0 hours	Day 1 Old medium 24 hours		Day 7 Fresh medium 0 hours	Day 8 Old medium 24 hours
Nominal concentration
of the test item [mg/L]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
10.0	1.6	1.11	69	0.866	0.745	86
Control	<LOQ	<LOQ   RR: 106%		<LOQ	<LOQ   RR: 99%
QC	RR: 99%			RR: 94%

Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account
% = percent of the initially measured concentration of the test item
LOQ = limit of quantification (0.005 mg/L of the test item)
QC = Quality control
RR = Recovery rate to nominal concentration of LOQ; mean value 106%

 

Sampling date	Day 08 Fresh medium 0 hours	Day 09 Old medium 24 hours		Day 14 Fresh medium 0 hours	Day 15 Old medium 24 hours
Nominal concentration
of the test item [mg/L]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
10.0	1.01	0.821	81	0.770	0.714	93
Control	<LOQ	<LOQ RR: 96%		<LOQ	<LOQ RR: 96%
QC	RR: 99%			RR: 101%

 

Sampling date	Day 15 Fresh medium 0 hours	Day 16 Old medium 24 hours
Nominal concentration
of the test item [mg/L]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Time weighted mean measured concentration
10.0	0.925	0.845	91	0.937
Control	<LOQ	<LOQ RR: 96%		<LOQ
QC	RR: 97%

 

Validity criteria fulfilled:

yes

Conclusions:

The overall effect threshold for effects of the test item under the test conditions was ≥ 0.937 mg/L
(NOEC) and > 0.937 mg/L (LOEC) based on the time weighted mean measured test item
concentration.

Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) of the test item Reaction mass of (3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphthol[2,1-b]furan and (3aS,5aR,9aR,9bS)-3a,6,6,9a- tetramethyldodecahydronaphthol[2,1-b]furan (batch number: 1005059223) was conducted according to OECD 211 (2012) from 2021-04-06 to 2021-05-12, with the definitive exposure phase from 2021-04-21 to 2021-05-12 at Noack Laboratorien.
Test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per saturated solution and control. At the start of the exposure the daphnids were less than 24 hours old. The study was carried out under semi-static conditions with a daily renewal of the test solutions. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, the number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.
The test was carried out as a limit test with a saturated solution of the test item (prepared with a nominal concentration of 10 mg test item /L), prepared with the test medium Elendt M4. The test was performed under semi-static conditions with a daily renewal of the test solutions in closed bottles without headspace. A limit test design was chosen because no effects on reproduction were expected at the saturated solution level.
The limit test item concentration and the control were analytically verified via GC-MS five times during the test in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7, 8, 14 and 15) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 9, 15 and 16). 
The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.
The effects on reproduction were evaluated based on the reproduction per female parent animal inserted at the start of the exposure. The effect values for the cumulative offspring per introduced parent are reported.

 

The overall effect threshold for effects of the test item under the test conditions was ≥ 0.937 mg/L (NOEC) and > 0.937 mg/L (LOEC) based on the time weighted mean measured test item concentration.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

mixture rules calculation

Adequacy of study:

supporting study

Study period:

From 16 december 2021 to 20 december 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

not applicable

Remarks:

(tested according to the Water Accommodated Fraction (WAF) approach, i.e. OECD technical guideline 23)

Principles of method if other than guideline:

The CHRONIC TOXICITY TO DAPHNIDS (21-DAY EL10) was determined using iSafeRat® calculation method adapted for a mixture of compounds with the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). This is an extension of the method published for acute exposure of nonpolar narcosis compounds (Bicherel and Thomas, 2021). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004b, 2007). The QSAR model is based on validated data for a training set of 30 chemicals derived from 21-day test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction.

GLP compliance:

no

Remarks:

Calculation method

Specific details on test material used for the study:

-Description: solid,
-Water Solubility:
ISOMER 1 : 2.32 mg/L (Kreatis 2021,a)
ISOMER 2 : 2.32 mg/L (Kreatis 2021,a)
-Mechanism of action:
ISOMER 1 :MechoA 1.1: non-polar narcosis (Bauer et al., 2018)
ISOMER 2 :MechoA 1.1: non-polar narcosis (Bauer et al., 2018)
Melting Point of the test item was measured to be 80°C (Firmenich, 2008).

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

Daphnia magna

Details on test organisms:

no information

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

Results from a test duration of 21 days only were used for Daphnia magna.

Post exposure observation period:

None

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The temperatures varied from approximately 20 to 23 °C.

pH:

Test results were taken from studies with measured pHs between 6 - 9.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations.

Salinity:

Not applicable

Conductivity:

no information

Nominal and measured concentrations:

Studies were used only where sufficient evidence was presented to determine that the stubstance was stable under test conditions (i.e. maintened within ± 20 % of the nominal) or, if not, the result was based on measured concentrations as geometric mean.

Details on test conditions:

Preferentially results from a flow-through test were used. However semi-static with daily renewal of test solutions and the control was accepted (preferably accompanied by analytical measurements over the study period). For suspected volatile substances only tests performed in closed vessels were accepted unless accompanying analytical monitoring proved such a design was not necessary.

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: (tested according to the Water Accommodated Fraction (WAF) approach, i.e. OECD technical guideline 23)

Basis for effect:

reproduction

Remarks on result:

not determinable

Details on results:

Each constituent of the test item falls within the applicability domain of the QSAR model used to determine their individual CHRONIC TOXICITY TO DAPHNIDS (21-DAY EC10). Moreover, each constituent of the test item acts with the same general MechoA.

Results with reference substance (positive control):

not applicable

Prior Analysis of the MechoA constituents of the test item.

The calculation method used in this study is based on toxic additivity principle. That means the toxic parts of each constituent are added up. Therefore the constituents considered within the mixture should act with a similar MechoA. The MechoA of the consituents are determined using the methodology described by Bauer et al. (2018) and reported in the Table below.

Constituents	MechoA	Description
Isomer 1	1.1	non-polar narcotic
Isomer 2	1.1	non-polar narcotic

Since the constituents of the test item act with the same general MechoA, the calculation method is directly applicable.

Validity criteria fulfilled:

yes

Remarks:

The test item falls within the applicability domain of the model and can therefore be considered a reliably prediction for chronic toxicity (21d-EC10) to daphnids.

Conclusions:

The CHRONIC TOXICITY TO DAPHNIDS (21-DAY EL10) of the test item has been determined using the iSafeRat® calculation method for mixtures tested according to the Water Accomodated Fraction (WAF) approach. Each constituent of the test item falls within the applicability domain of the QSAR model used to determine their individual CHRONIC TOXICITY TO DAPHNIDS (21-DAY EC10). Moreover, each constituent of the test item acts with the same general MechoA. Therefore the calculation method is directly applicable and the final result for the test item can be considered valid for use in risk assessment and classification and labelling. Therefore, the final result for the test item is considered as reliable without restrictions. The result is valid for use in risk assessment and classification and labelling.
The CHRONIC TOXICITY TO DAPHNIDS (21-DAY EL10) of the test item tested according to the WAF method was predicted as a loading rate of 2.0E-01 mg/L.

Executive summary:

A calculation method was used to assess the CHRONIC TOXICITY TO DAPHNIDS (21-DAY EL10) of the test item, a multiconstituent substance, tested according to the Water Accommodated Fraction (WAF) approach. This calculation method predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the Guideline for Testing of Chemicals No. 211, "Daphnia magna Reproduction Test" (OECD, 2012) adapted for testing of a mixture using the WAF method. The criterion predicted was the EL10 (10% Effective Loading rate of the mixture), a statistically derived loading rate which is expected to cause an effect of 10% on reproducibility within a period of 21 days.

The CHRONIC TOXICITY TO DAPHNIDS (21-DAY EL10) was determined using iSafeRat® calculation method adapted for a mixture of compounds with the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). This is an extension of the method published for acute exposure of nonpolar narcosis compounds (Bicherel and Thomas, 2021). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004b, 2007). The QSAR model is based on validated data for a training set of 30 chemicals derived from 21-day test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction.

 

Each constituent of the test item falls within the applicability domain of the QSAR model used to determine their individual CHRONIC TOXICITY TO DAPHNIDS (21-DAY EC10). Moreover, each constituent of the test item acts with the same general MechoA. Therefore the calculation method is directly applicable and the final result for the test item can be considered valid for use in risk assessment and classification and labelling. Therefore, the final result for the test item is considered as reliable without restrictions. The result is valid for use in risk assessment and classification and labelling.

The CHRONIC TOXICITY TO DAPHNIDS (21-DAY EL10) of the test item tested according to the WAF method was predicted as a loading rate of 2.0E-01 mg/L.

Description of key information

OECD Guideline 211, GLP, key study, validity 1:

NOEC reproduction (Daphnia magna) = ≥ 0.937 mg/L based on time weighted mean measured concentrations

LOEC reproduction (Daphnia magna) = > 0.937 mg/L based on time weighted mean measured concentrations

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 0.937 mg/L

Additional information

To assess the long-term toxicity of the registered substance to aquatic invertebrates, two data point are available

The first data (NOACK, 2021) is assessed as the key study and is an experimental study. In this reproduction test with Daphnia magna, the effects on the reproduction capacity and other sub-lethal effects of a saturated solution of the test item ( prepared with a nominal concentration of 10 mg test item/ L)  were determined at the test facility according to OECD Guideline 211 with GLP compliance. This study was performed over a period of 21 days under semi-static and closed system without headspace, with a daily renewal of the test solutions. The limit test item concentration and the control were analytically verified via GC-MS five times during the test in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7, 8, 14 and 15) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 9, 15 and 16). The effects on reproduction were evaluated based on the reproduction per female parent animal inserted at the start of the exposure. The effect values for the cumulative offspring per introduced parent are reported. The overall effect threshold for effects of the test item under the test conditions was ≥ 0.937 mg/L (NOEC) and > 0.937 mg/L (LOEC) based on the time weighted mean measured test item concentration.

The second value (KREATiS, 2021) is assessed as a supporting datapoint and is a calculation method. This calculation method was used to assess the CHRONIC TOXICITY TO DAPHNIDS (21-DAY EL10) of the test item, a multiconstituent substance, tested according to the Water Accommodated Fraction (WAF) approach. The CHRONIC TOXICITY TO DAPHNIDS (21-DAY EL10) was determined using iSafeRat® calculation method adapted for a mixture of compounds with the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). This is an extension of the method published for acute exposure of nonpolar narcosis compounds (Bicherel and Thomas, 2021). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004b, 2007). Each constituent of the test item falls within the applicability domain of the QSAR model used to determine their individual CHRONIC TOXICITY TO DAPHNIDS (21-DAY EC10). Moreover, each constituent of the test item acts with the same general MechoA. Therefore the calculation method is directly applicable and the final result for the test item can be considered valid for use in risk assessment and classification and labelling. Therefore, the final result for the test item is considered as reliable without restrictions. The result is valid for use in risk assessment and classification and labelling. The CHRONIC TOXICITY TO DAPHNIDS (21-DAY EL10) of the test item tested according to the WAF method was predicted as a loading rate of 2.0E-01 mg/L.