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

09 March 2021 to 20 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev.4, Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414 (11/07/00)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals"

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

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

Specific details on test material used for the study:

Storage condition : dry area, protect 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:

Sampling : the test item concentrations were analytically verified via GC-MS four times during the test (Once within a period of 7 days) in the fresh media at the start of an exposure- renewal interval (0 hours; on test days 0, 7, 14 and 19) as well as in the old media at the end of an exposure- renewal interval (24 hours; on test days 1, 8 15 and 20) in all WAFs and in the control.
All concentration levels and the control were analyzed via GC-MS at 4 times during the test (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.
At the start of the renewal intervals, samples of the fresh media monitoring were taken after preparation of all loading rates 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:

Water accommodated fractions (WAF) were prepared because the test item is a UVCB substance with compounds of different water solubility. Aqueous media were prepared by mixing the
test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. Five water accommodated fractions (WAF) were prepared accommodated fractions 24 ± 1 hours before the start of the exposure and each renewal day with nominal loading rates of the test item in the range of 0.0512 to 2.00 mg/L set up in a geometric series with a factor of 2.5: 0.0512 - 0.128 - 0.320 - 0.800 - 2.00 mg/L.
For the loading rate 0.800 and 2.00 mg/L an appropriate amount of the test item was weighed out and transferred into a glass bottle with an appropriate amount of the dilution water (see Table 2). The dispersions were stirred for 24 ± 1 hours at approximately 1100 rpm with a magnetic stirrer at room temperature. The dispersion was removed by siphoning from the middle of the water column and was centrifuged (17000 g for 20 minutes at 20 ± 1 °C) to separate undissolved test item. The WAFs were checked via laser beam (Tyndall effect) for undissolved test item. No presence of undissolved test item during the test was observed. The resulting water accommodated fractions (WAFs) were used in the test. The loading rates 0.0512 – 0.128 – 0.320 mg/L were prepared by dilution from 0.800 mg/L. The direct application of these loading rates was not possible.

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:

Elendt m4, according to OECD 211

Limit test:

no

Total exposure duration:

21 d

Hardness:

measured 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
Highest
loading rate 240 263 240 269 247 257
Control 266 261 266 243 292 265
The total hardness was above 140 mg/L as CaCO3.

Test temperature:

Measured temperature between 19.8 and 21.1°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 at test start and between 7.61 et 8.09 at test end
The pH in the highest loading rate was between 7.42 and 7.65 at test start and between 7.44 and 7.83 at test end. 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 8.65 and 9.11 at test start and between 6.91 et 7.86 at test end
The pH in the highest loading rate was between 8.25 and 8.70 at test start and between 6.00 and 7.78 at test end. The dissolved oxygen concentration was above 3 mg/L.

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Five WAFs were prepared and tested at nominal loading rates (spaces by a factor of 2.5) : 0.0512 - 0.128 - 0.320 - 0.800 - 2.00 mg/L.
Measured concentrations, see table in "Any other information on results incl. tables".

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.
- Neonates: First appearance of juveniles was checked daily. The number of neonates (alive and dead progeny) was counted and dates of release were recorded once a day from the first day of appearance of neonates until the end of the test. 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 dates of observation were recorded.
- 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

Key result

Duration:

21 d

Dose descriptor:

other: LOEL

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

other: NOEL

Effect conc.:

0.128 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.628 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.268 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

other: LL20

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LL50

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LL100

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: NOEL

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LOEL

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Reproductive output: The number of juveniles in all replicates of the control and the test groups was counted and recorded every day. The average number of living juveniles at the end of the test after 21 days per surviving parental daphnid was 115 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per surviving parent animal in the control was met.
Accidental and inadvertent mortality as defined in the OECD test guideline were not observed.
Since the Cochran-Armitage trend test revealed no significant increasing trend (p ≤ 0.05) in adult mortality, the evaluation was done based on the cumulative offspring per survived (female) parent.
A statistically significant reduction of the reproductive output in comparison to the reproductive
output in the control was determined at the nominal loading rates of 0.320 to 2.00 mg/L. Therefore, the LOEL was determined to be 0.320 mg/L and the NOEL 0.128 mg/L for the cumulative offspring per survived parent.
The determination of the EL10- and EL 50-values and their 95% confidence limits was carried out by point estimates from the 3-parametric normal CDF. The EL10 for the test item was 0.268 mg/L (Cl: 0.194 – 0.371) and the EL50 0.628 mg/L (Cl: 0.425 – 0.928) per survived parent.
Adult mortality of 10% after 21 days was observed in the nominal loading rates 0.128 to 2.00 mg/L. In the control and the loading rate 0.0512 mg/L no adult mortality was observed. The statistical evaluation of the mortality was determined with Fisher’s exact binominal test with Bonferroni correction. The NOEL and LOEL were determined to be ≥ 2.00 and > 2.00 mg/L.
The intrinsic rate of population increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations it will be zero. For growing populations it will be positive and for shrinking populations it will be negative.
The first appearance of living juveniles was observed between days 7 and 12 at the introduced parental daphnids of the control and the nominal loading rates of 0.0512 to 2.00 mg/L of the test item.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

The NOEL and LOEL for the reproduction and the adult mortality 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 Williams multiple sequentially t-test for reproduction. The Fisher’s
exact binominal test with Bonferroni correction was used for adult mortality. P-values of the normality and the variance homogeneity test were 0.01.
For the effects on reproduction, the juveniles per survived 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.

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 115 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
Highest loading rate	7.42	7.83	7.58	7.68	7.65	7.44
Control	7.43	8.02	8.11	7.61	7.49	8.09

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
Highest loading rate	8.25	6.00	8.70	7.39	8.59	7.78
Control	8.65	6.91	8.93	7.86	9.11	7.25

 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
Highest loading rate	240	263	240	269	247	257
Control	266	261	266	243	292	265

 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
Highest loading rate	19.9	20.2	20.2	20.8	20.5	20.4
Control	19.8	20.1	20.4	21.1	19.6	20.1

 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
2.00 0.800 0.320 0.128	5.3 41.1 95.1 124	3.64 9.52 14.5 18.1	68.3 23.2 15.2 14.6
0.0512	106	10.9	10.3
control	115	21.6	18.8

 

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
2.00 0.800 0.320 0.128	0 0 0 10	0 0 0 10	0 0 0 10
0.0512	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 (%)
2.00 0.800 0.320 0.128 0.0512 Control	0.148±0.0485 0.321±0.0155 0.363±0.0170 0.362±0.0200 0.369±0.0218 0.372±0.0216	32.9 4.8 4.7 5.5 5.9 5.8

 

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
2.00 0.800 0.320 0.128 0.0512 Control	10 8 8 8 7 8	12 9 9 9 8 8	10 8 8 8 8 8	8 8 8 8 8 8	8 8 7 7 8 8	10 8 8 8 7 8	9 10 9 8 8 9	8 8 9 8 7 8	8 8 8 8 9 8	8 8 8 - 8 8	9.6 8.8 8.7 8.5 8.3 8.6

 

Stillborn Juveniles and Aborted Eggs produced by the Survived Parental Daphnids after 21 Days

 

Nominal loading rate of the test item (mg/L)	Number of		Total no.
	Stillborn juveniles	Aborted eggs	Ʃ
2.00 0.800 0.320 0.128 0.0512 Control	316 457 63 24 6 15	39 30 20 13 2 0	355 487 83 37 8 15

 

 

 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
2.00 0.800 0.320 0.128 0.0512 Control	4.0 4.5 4.5 4.0 4.5 4.5	4.0 4.5 - 4.5 4.5 4.5	4.0 - 4.5 4.5 4.0 4.5	4.0 4.5 4.5 4.5 4.5 4.5	3.5 4.0 3.5 4.5 4.5 4.0	4.0 3.5 4.5 4.5 4.5 4.5	- 4.0 3.5 4.5 4.5 4.5	3.5 4.5 4.5 4.5 4.5 4.5	4.0 4.0 4.5 4.5 4.5 4.0	4.0 4.5 4.5 - 4.0 4.5	3.89 4.22 4.28 4.44 4.40 4.40	9 9 9 9 10 10	3.8 5.8 5.4 6.7 6.4 5.7	0.42 0.64 0.60 0.74 0.64 0.57

N = number of the survived parental female daphnids

- = not applicable, due to the mortality of the prental daphnid(s)

 

Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles per Survived Parental Daphnids

 

Nominal loading rate of the test item (mg/L)	Number of juveniles
	Dead /above	Alive
2.00 0.800 0.320 0.128 0.0512 Control	316/39 457/30 63/20 24/13 6/2 15/0	52 408 913 1115 1061 1149

Dead = aborted eggs + stillborn juveniles

Total = dead + alive juveniles

* related to the total number of juveniles

Analytical results : 

The test item concentrations of the test item were analytically verified via GC-MS four times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7, 14 and 19) as well as in the old media at the end of an exposurerenewal
interval (24 hours; on test days 1, 8, 15 and 20) in all WAFs and in the control. 

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	Reaction mass of (3aR*,5aS*,9aS*,9bR*)-3a,6,6,9a- Tetramethyldodecahydronaphtho[2,1-b]furan and (3aR*,5aS*,9aS*,9bS*)-3a,6,6,9a- Tetramethyldodecahydronaphtho[2,1-b]furan
of the test item [mg/L]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
2.00	0.751	0.652	87	0.570	0.517	91
0.800	0.401	0.362	90	0.493	0.446	90
0.320	0.159	0.144	90	0.145	0.138	95
0.128	0.0602	0.0538	89	0.0587	0.0554	94
0.0512	0.0264	0.0243	92	0.0230	0.0207	90
Control	<LOQ	<LOQ   RR: 102%		<LOQ	<LOQ   RR: 110%
QC	RR: 100%			RR: 111%

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 14 Fresh medium 0 hours	Day 15 Old medium 24 hours		Day 19 Fresh medium 0 hours	Day 20 Old medium 24 hours
Nominal concentration	Reaction mass of (3aR*,5aS*,9aS*,9bR*)-3a,6,6,9a- Tetramethyldodecahydronaphtho[2,1-b]furan and (3aR*,5aS*,9aS*,9bS*)-3a,6,6,9a- Tetramethyldodecahydronaphtho[2,1-b]furan
of the test item [mg/L]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
2.00	0.984	0.872	89	0.551	0.486	88
0.800	0.401	0.384	96	0.368	0.310	84
0.320	0.0534	0.0552	103	0.136	0.146	107
0.128	0.0211	0.0218	103	0.0664	0.0610	92
0.0512	0.00868	0.0134	154	0.0285	0.0240	84
Control	<LOQ	<LOQ RR: 107%		<LOQ	<LOQ RR: 107%
QC	RR: 106%			RR: 105%

Validity criteria fulfilled:

yes

Conclusions:

The overall effect threshold for effects of the test item under the test conditions was 0.128 mg/L
(NOEL) and 0.320 mg/L (LOEL) based on the nominal loading rates of the test item

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-Tetramethyldodecahydronaphtho[2,1-b]furan and (3aR*,5aS*,9aS*,9bS*)-3a,6,6,9a-tramethyldodecahydronaphtho[2,1-b]furan (batch number:1005147237) was conducted according to OECD 211 (2012) from 2021-03-09 to 2021-03-31, with
the definitive exposure phase from 2021-03-10 to 2021-03-31 at Noack Laboratorien.
Test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per WAF loading rate 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 aqueous media were prepared following OECD guidance document No. 23 (2019), which is specifically developed for aquatic toxicity testing of UVCBs, by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon ing.
This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 0.0512 - 0.128 - 0.320 - 0.800 - 2.00 mg/L (separation factor 2.5).
The WAFs were checked for any undissolved or emulsified material by Tyndall effect, which was negative.
The test item concentrations were analyzed via GC-MS four times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours) as well as in the old media at the end of an exposure-renewal interval (24 hours) in all WAFs and in the control. Details of the analytical method and the method validation are presented in section 14 and section 17.
As the test item is a UVCB substance, all evaluations are based on the nominal loading rates of the test item.
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 Cochran-Armitage trend-test revealed no significant trend in mortality. Thus, according to OECD 211 it is required to report the effect values for the cumulative offspring per survived parent. All reasonable efforts were taken to produce a solution of all soluble
components of the test item in test media. Since the test item is a multicomponent mixture (UVCB), the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. According to OECD guidance document No. 23 (2019), for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or loading rate (for mixtures).

Effect values	Reaction mass of (3aR*,5aS*,9aS*,9bR*)-3a,6,6,9a- Tetramethyldodecahydronaphtho[2,1-b]furan and (3aR*,5aS*,9aS*,9bS*)-3a,6,6,9a- Tetramethyldodecahydronaphtho[2,1-b]furan [mg/L]
EL10 Reproduction (survived)	0.268 (Cl: 0.194 – 0.371)
EL50 Reproduction (survived)	0.628 (Cl: 0.425 – 0.928)
LOEL Reproduction (survived)	0.320
NOEL Reproduction (survived)	0.128
LL20 Adult mortality after 21 days	>2.00
LL50 Adult mortality after 21 days	>2.00
LL100 Adult mortality after 21 days	>2.00
LOEL Adult mortality after 21 days	>2.00
NOEL Adult mortality after 21 days	>2.00

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

mixture rules calculation

Adequacy of study:

supporting study

Study period:

From 02 November 2021 to 22 november 2022

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

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. This small difference is not expected to contribute to the variability of the values found in experimental data.

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:

EL10

Effect conc.:

0.23 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
Isomer 3 (impurity)	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 2.3E-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 2.3E-01 mg/L.

Description of key information

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

21d-EL10 reproduction (Daphnia magna) : 0.268 mg/L based on nominal loading rates.

21d-EL50 reproduction (Daphnia magna) : 0.628 mg/L based on nominal loading rates.

LOEL reproduction (Daphnia magna) : 0.320 mg/L based on nominal loading rates.

NOEL reproduction (Daphnia magna) : 0.128 mg/L based on nominal loading rates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

0.268 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 five water accommodated fractions (WAF) with nominal loading rates of 0.0512 - 0.128 - 0.320 - 0.800 - 2.00 mg/L of the registered substance 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 concentrations of the test substance were analytically verified via GC-MS in fresh media on days 0, 7, 14, 19 (0 hours) and in old media on days 1, 8, 15, 20 (24 hours) in all nominal loading rates and the control. The effects on reproduction were evaluated based on the reproduction per inserted at the start of the exposure. No significant trend in mortality was observed. Therefore, the evaluation has to be based on the cumulative offspring per survived parent. The effects on reproduction were evaluated based on the reproduction per female parent animal inserted at the start of the exposure. The Cochran-Armitage trend-test revealed no significant trend in mortality. Thus, according to OECD 211 it is required to report the effect values for the cumulative offspring per survived parent based on the nominal loading rates of the test substance. Therefore, the LOEL was determined to be 0.320 mg/L and the NOEL 0.128 mg/L for the cumulative offspring per survived parent. The EL10 for the test item was 0.268 mg/L (Cl: 0.194 – 0.371) and the EL50 0.628 mg/L (Cl: 0.425 – 0.928) per survived parent.

 

The second data (QSAR-KREATiS, 2020) is assessed as 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. 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) of the test item tested according to the WAF method was predicted as a loading rate of 2.3E-01 mg/L.