Reaction mass of (2Z)-5-[(1R,3R,6S)-2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl]-2-methyl-2-penten-1-ol and (2Z)-2-methyl-5-[(1S,2R,4R)-2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl]-2-penten-1-ol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 February 2020 to 5 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 29-30-31 May 2018 / Signature date: 15 November 2018

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test solutions (from a replicate of each test concentration with algae dedicated exclusively to chemical analyses) at the start and the end of the test. Chemical analyses were performed on the two major constituents of the test item and used as tracers to provide an indication of the concentration of dissolved organic material in the WAFs and stability.

Vehicle:

no

Details on test solutions:

- Preparation of Water Accommodated Fractions (WAFs): The study was carried out using WAFs. The WAFs were prepared under closed conditions and by slow-stirring. The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each test vessel and 5.0 L to 5.4 L of test water (depending on the brim capacity of the bottles) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 11 days of gentle stirring in the dark at room temperature, the WAFs were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL of solution were discarded via the drain port. Then the WAFs were directly added into test vessels containing a fixed amount of inoculum (5.103 cells/mL per vessel) that were immediately sealed after filling with a minimum headspace. At the start of the test, the test solutions in all test vessels were observed to be clear and colourless. The Tyndall effect (checked via laser beam) was negative in all treatments. The pH of the WAFs was slightly adjusted to be in the range of 8.1 ± 0.2.
- Test concentrations: Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following loading rates (spaced by a factor of approximately 3.20): 0.31, 1.0, 3.1, 10.0 and 32.0 mg/L.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Species: Pseudokirchneriella subcapitata, CCAP 278/4
- Origin: Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Reason for selection: This system is a unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
- Stock culture: Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.
- Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1.104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

The temperature in the incubator was situated between 21.1 and 23.0°C throughout the test (average value: 22.9°C).

pH:

See Table 6.1.5/1 in "Any other information on results incl. tables"
pH not varying by more than 1.5 units at the end of the test in the control

Dissolved oxygen:

No data

Salinity:

Not applcable

Conductivity:

No data

Nominal and measured concentrations:

- Nominal Loading Rates: 0.31, 1.0, 3.1, 10.0 and 32.0 mg/L.
- Measured concentrations: See tables 6.1.5/4 and 5 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test type: A static test was performed.
- Test vessels: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Cell concentration An initial cell density of 5.103 cells/mL using the exponentially growing pre-culture.
- Replicates: 6 controls and 3 replicates of each test concentration for counting. Moreover, additional replicates of each treatment with algae were prepared for chemical analyses (destructive samples) in order to determine maintenance of actual concentrations.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
Original medium from OECD TG 201
Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water to insure a satisfactory CO2 supply for the algal growth (for all treatments and inoculum suspension): 7 mL of NaHCO3 were added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg/L.

OTHER TEST CONDITIONS
- Test environment: Controlled environment cabinet (23°C ± 2°C); vessels were distributed randomly in the incubator and redistributed over the test at t=24h and t=48h. During incubation, the algal cells were kept in suspension by continuous magnetic stirring (250 rpm).
- Light intensity and quality: Continuous illumination with a light intensity of 4,440-8,880 lux and did not vary more than ± 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Cell densities: Cell numbers were counted daily by microscope using a counting chamber. The software ToxRat® Professional was used to perform statistical analyses. Complementary statistical analyses were performed using another statistical spreadsheet (using Excel®) for the validity criteria of the study.
- pH: At the start and the end of the test in one vessel per concentration and the control (same vessel at t=0h and t=72h).
- Temperature of medium: Measured continuously in the growth chamber, over the study period.
- Light intensity: Light intensity was measured once (t=0h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

RANGE-FINDING STUDY:
A range-finding test was conducted to determine the range of concentrations for the definitive test. This range-finding test was carried out using WAFs of the test item over a range of nominal loading rate of 0.32, 1.0, 3.2, 10.0 and 50.0 mg/L and to a control. The WAFs were in the dark under closed conditions and by slow-stirring. The mixing vessels were graduated cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each mixing vessel and 5200 mL to 5700 mL of test water (original medium of OECD TG 201) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. [For the preparation of the highest loading rate (50 mg/L), the test item was weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing vessel was then carefully filled with the remaining volume of test water to obtain 5.7 L of test water]. Then the mixing vessels were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 22.5 hours of gentle stirring in the dark and at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were discarded via the drain port. Then the WAFs were directly added into test vessels containing a fixed amount of inoculum (5.103 cells/mL per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, test solutions in test vessels were observed to be clear and colourless in all loading rates. The test was carried out without adjustment of the pH. Test solutions in mixing and test vessels were checked via laser beam; the Tyndall effect was negative in all treatments. Based on chemical analysis, low recoveries of the test substance were observed for both constituents. A complementary test was performed and showed that additional time was required to reach the expected water solubility of each constituent in Original medium of OECD TG 201, especially at the highest loading rates. This is why it was decided to use a slow stirring period of approximately 11 days for the final test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

3.341 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1.808 - 4.479 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

6.337 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 4.831 - 7.727 mg/L

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Light intensity: The mean light intensity was 5445 lux (range: 5225-5605 lux).
- Analytical results: See tables 6.1.5/4 and 5 in "Any other information on results incl. tables". The analytical results of this test showed that WAFs were overall stable throughout the test period. Indeed, analytical monitoring of the two major constituents of the test item revealed that their concentrations were satisfactorily maintained within or very close to ± 20% of the initial concentration between the start and the end of the test. Besides, these concentrations were overall satisfactorily maintained within ± 20% of their expected nominal concentrations throughout the test. Therefore, and since the test item was a multi-constituent substance, the results were based on nominal loading rates and as Effective Loading Rate 50, according to the OECD No. 23.
- Biological results: See tables 6.1.5/2 and 3 in "Any other information on results incl. tables".

Results with reference substance (positive control):

On February 13, 2020 (KA20-001; most recent test), the 72h-EC50 was 0.961 mg/L for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-ErC50: 0.65 mg/L to 1.73 mg/L).

Table 6.1.5/1: pH-values during the final test

	Nominal concentration(mg test item/L)*
	Control	0.31	1.0	3.1	10.0	32.0
Start t=0 h	8.34	8.27	8.28	8.10	8.18	8.18
End t=72 h	9.51	9.35	9.37	9.18	8.35	8.25

* WAF prepared at the given loading rate.

Table 6.1.5/2: Algal cell densities during the final test (expressed as density of algal cells/mL x 10^4)

	Replicate	Nominal concentration  (mg test item/L)*
		Control	0.31	1.0	3.1	10.0	32.0
t=24 h	1	1.2	1.6	0.4	1.6	0.8	0.4
	2	1.6	3.2	1.2	1.2	0.0	0.4
	3	1.2	2.0	1.6	1.2	0.8	0.4
	4	2.8
	5	2.0
	6	1.6
	Mean	1.7	2.3	1.1	1.3	0.5	0.4
	Std. Dev.	0.60	0.83	0.61	0.23	0.46	0.00
t=48 h	1	10.8	13.6	7.6	9.2	2.0	2.4
	2	8.4	13.6	8.4	8.0	0.4	2.0
	3	15.6	14.0	10.0	9.2	0.8	0.8
	4	13.6
	5	10.0
	6	16.0
	Mean	12.4	13.7	8.7	8.8	1.1	1.7
	Std. Dev.	3.13	0.23	1.22	0.69	0.83	0.83
t=72 h	1	26.8	34.4	24.0	28.0	1.2	0.8
	2	28.0	35.2	28.0	22.4	0.8	1.6
	3	34.4	36.0	30.4	21.2	1.2	0.8
	4	36.0
	5	32.4
	6	36.8
	Mean	32.4	35.2	27.5	23.9	1.1	1.1
	Std. Dev.	4.17	0.80	3.23	3.63	0.23	0.46

* WAF prepared at the given loading rate.

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls and 3 replicates of each test concentration.

Std. Dev.: standard deviation.

Table 6.1.5/3: Mean specific growth rate in P. subcapitata during the final test

	Replicate	Nominal concentration  (mg test item/L)*
		Control	0.31	1.0	3.1	10.0	32.0
t=0 h - t=24 h	1	0.875	1.163	-0.223	1.163	0.470	-0.223
	2	1.163	1.856	0.875	0.875	-1.609	-0.223
	3	0.875	1.386	1.163	0.875	0.470	-0.223
	4	1.723
	5	1.386
	6	1.163
	Mean	1.198	1.469	0.605	0.971	-0.223	-0.223
	Std. Dev.	0.3229	0.3538	0.7316	0.1661	1.2006	0.0000
	% Inh.	-	-22.6	49.5	18.9	118.6	118.6
t=0 h - t=48 h	1	1.536	1.652	1.361	1.456	0.693	0.784
	2	1.411	1.652	1.411	1.386	-0.112	0.693
	3	1.720	1.666	1.498	1.456	0.235	0.235
	4	1.652
	5	1.498
	6	1.733
	Mean	1.592	1.656	1.423	1.433	0.272	0.571
	Std. Dev.	0.1301	0.0084	0.0694	0.0403	0.4036	0.2944
	% Inh.	-	-4.1	10.6	10.0	82.9	64.1
t=0 h - t=72 h	1	1.327	1.410	1.290	1.342	0.292	0.157
	2	1.342	1.418	1.342	1.267	0.157	0.388
	3	1.410	1.426	1.369	1.249	0.292	0.157
	4	1.426
	5	1.390
	6	1.433
	Mean	1.388	1.418	1.334	1.286	0.247	0.234
	Std. Dev.	0.0442	0.0076	0.0400	0.0491	0.0780	0.1334
	% Inh.	-	-2.2	3.9	7.3	82.2	83.2

* WAF prepared at the given loading rate.

Values were extracted from the computer program ToxRat.

% Inh.: %Inhibition of growth rate relative to the control determined by ToxRat.

Table 6.1.5/4: Concentrations of the test item (mg/L) in test water in the final test - Constituent 1

Nominal concentration* (mg/L)	Expected nominal concentration in consituent 1 (mg constituent 1/L)	Measured concentration in consituent 1			Geometric mean measured concentrations
		Start (t=0 h)	End (t= 72h)	Relative  loss to initial value (t=0h - t=72h) (%)	mg/L	% nominal
Control	0	Abs.	Abs.	N.A.	N.A.	N.A.
0.31	0.17	Pres.^a	Pres.^a	N.A.	0.10	59
1.0	0.54	0.62	0.48	22	0.55	102
3.1	1.69	1.54	1.58	-3	1.56	92
10.0	5.45	5.15	5.60	-9	5.37	99
32.0	saturation ca. 5.7^b	4.67	5.52	-18	5.08	89

* WAF prepared at the given loading rate

N.A.: not applicable

% = Percent of expected nominal concentration in Constituent 1.

^aPres= Presence: concentrations below the LOQ (0.20 mg/L) but above the LOD (0.06 mg/L). Where measured concentrations were below the LOQ (0.20 mg/L) but above the LOD (0.06 mg/L), such concentrations were considered to be the half of the quantification limit, according to the study plan ( => LOQ/2 = 0.10 mg/L).

Abs= Absence: concentrations below the LOQ (0.20 mg/L) and the LOD (0.06 mg/L).

^bBased on the water solubily test result (OECD 105; LPL, 2020).

Table 6.1.5/5: Concentrations of the test item (mg/L) in test water in the final test - Constituent 2

Nominal concentration* (mg/L)	Expected nominal concentration in consituent 2 (mg constituent 2/L)	Measured concentration in consituent 2			Geometric mean measured concentrations
		Start (t=0 h)	End (t= 72h)	Relative loss to initial value (t=0h - t=72h) (%)	mg/L	% nominal
Control	0	Abs.	Abs.	N.A.	N.A.	N.A.
0.31	0.08	Pres.^a	Pres.^a	N.A.	0.10	125
1.0	0.26	0.31	0.25	19	0.28	108
3.1	0.79	0.74	0.78	-5	0.76	96
10.0	2.56	2.54	2.79	-10	2.66	104
32.0	saturation ca. 3.6^b	3.33	3.87	-16	3.59	100

* WAF prepared at the given loading rate

N.A.: not applicable

% = Percent of expected nominal concentration in Constituent 2.

^aPres= Presence: concentrations below the LOQ (0.20 mg/L) but above the LOD (0.06 mg/L). Where measured concentrations were below the LOQ (0.20 mg/L) but above the LOD (0.06 mg/L), such concentrations were considered to be the half of the quantification limit, according to the study plan (=> LOQ/2 = 0.10 mg/L).

Abs= Absence: concentrations below the LOQ (0.20 mg/L) and the LOD (0.06 mg/L).

^bBased on the water solubily test result (OECD 105; LPL, 2020).

Validity criteria fulfilled:

yes

Remarks:

Cell density in controls: 65-fold increase within 72h; The mean CV for section-by-section specific growth rates in the control was 22.0% in 72h. The CV of average specific growth rates during the whole test period in replicate control cultures was 3.2%

Conclusions:

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 72h-ErL50 and 72h-ErL10 for growth rate were determined to be 6.337 mg/L and 3.341 mg/L, respectively. The 72h-NOELR and 72h-LOELR values for growth rate were 3.100 mg/L and 10.000 mg/L.

Executive summary:

A GLP study was performed to assess the test item for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline 201, referenced as EU Method C.3 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item in closed and static conditions over a range of nominal loading values of 0.31, 1.0, 3.1, 10.0 and 32.0 mg/L and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The concentrations of the test item, represented by analytical follow-up of the two main constituents, were determined at the start and the end of the test.

Analytical monitoring of the two major constituents of the test item revealed that their concentrations were satisfactorily maintained within or very close to ± 20% of the initial concentration between the start and the end of the test, suggesting that WAFs were overall stable throughout the test.

Under the experimental conditions and based on nominal loading rates, the 72h-ErL50 and 72h-ErL10 for growth rate were determined to be 6.337 mg/L and 3.341 mg/L, respectively. The 72h-NOELR and 72h-LOELR values for growth rate were 3.100 mg/L and 10.000 mg/L.

Description of key information

OECD Guideline 201, EU Method C.3, GLP, Key study, validity 1:

72h-ErL50 (Pseudokirchneriella subcapitata) = 6.337 mg/L (95% CL: 4.831 - 7.727 mg/L)

72h-ErL10 (Pseudokirchneriella subcapitata) = 3.341 mg/L (95% CL: 1.808 - 4.479 mg/L)

Key value for chemical safety assessment

EC50 for freshwater algae:

6.337 mg/L

EC10 or NOEC for freshwater algae:

3.341 mg/L

Additional information

One key study is available to assess the toxicity of the registered substance to aquatic algae Pseudokirchneriella subcapitata.

This study (LPL, 2020) was performed according to OECD Guideline 201 and EU Method C.3 with GLP compliance. Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the registered substance in closed and static conditions over a range of nominal loading values of 0.31, 1.0, 3.1, 10.0 and 32.0 mg/L and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The concentrations of the test item, represented by analytical follow-up of the two main constituents, were determined at the start and the end of the test. Analytical monitoring of the two major constituents of the test item revealed that their concentrations were satisfactorily maintained within or very close to ± 20% of the initial concentration between the start and the end of the test, suggesting that WAFs were overall stable throughout the test. Under the experimental conditions and based on nominal loading rates, the 72h-ErL50 and 72h-ErL10 for growth rate were determined to be 6.337 mg/L and 3.341 mg/L, respectively. The 72h-NOELR and 72h-LOELR values for growth rate were 3.100 mg/L and 10.000 mg/L.