(4E)-4-(3,3,4-trimethylcyclopentylidene)butanal

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 23 September 2019 to 17 October 2019

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:

Inspection dates: 29-30-31 May 2018 / Signature date: 15 November 2018

Specific details on test material used for the study:

- Appearance: From very light yellow to yellow liquid
- Storage condition of test material: Room temperature. Keep only in the original container in an appropriate place.
- Water solubility: 50.3 mg/L at 20°C (LPL 2020, OECD 105/EU A.6, slow-stirring method)
- Vapour pressure: 5.0 Pa at 20°C and 8.7 Pa at 25°C (LPL 2020, OECD 104/EU A.4, effusion method, Knudsen cell)
- Log Kow: 4.3 at 25°C (LPL 2020, OECD 123/EU A.23, slow-stirring method)

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration with algae dedicated exclusively to chemical analyses) at the start and every day thereafter until the end of the test.
- Samples preparation: Specimen were injected directly after a dilution by a factor of two with acetonitrile (500 µL sample + 500 µL acetonitrile). If the sample concentrations were too high and not included in the concentration range of the calibration, the samples were diluted appropriately with a test water/acetonitrile (50/50 v/v) mixture.

Vehicle:

no

Details on test solutions:

- Preparation of stock and test solutions: The mixing vessel was a cylindrical glass bottle sealed with a screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and 1.2 L test water was added. Then an excess of the test item (approx. 1.05 g) was carefully added directly to the surface of the test water. The mixing vessel was thereafter 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 23 hours of gentle stirring in the dark at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the remaining stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary based on the measured concentration of the stock solution (≈ 50.58 mg/L) and a fixed amount of inoculum to obtain the required test concentrations in the test vessels and a cell density of 5.103 cells/mL per vessel. At the start of the test, the test solutions in test vessels were observed to be clear and colourless.
- Test concentrations: Based on the results of a range-finding test, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water and inoculum to obtain the following nominal concentrations (spaced by a factor of approximately 3.00): 0.30, 0.9, 2.8, 8.3 and 25.0 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:

350 mg/L

Test temperature:

The temperature of the test medium was situated between 23.0 and 24.1°C throughout the test.
(temperature: 23°C ± 2°C, constant within 2°C)

pH:

The pH of the controls was 8.25 at the start of the test and 9.51 at the end of test.
(pH: not varying by more than 1.5 units at the end of the test in the control)

Dissolved oxygen:

no data

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

- Nominal concentrations: 0.30, 0.90, 2.80, 8.30 and 25 mg/L
- Geometric mean measured concentrations after 48h: 0.31, 0.82, 2.61, 8.11 and 24.85 mg/L
- Geometric mean measured concentrations after 72h: 0.20, 0.75, 2.27, 7.90 and 24.38 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
- Type (delete if not applicable): closed
- Initial cells density: An initial cell density of 5.103 cells/mL using the exponentially growing pre-culture.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
Original medium from OECD TG 201. The pH of this solution was approximately in the range of 8.1 ± 0.2. 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
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.
Continuous illumination (the mean light intensity was 5647 lux (range: 5320-5871 lux)).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
A range-finding test was conducted between September 2 and 6, 2019 to determine the range of concentrations for the definitive test. The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and approx. 1 L test water was added. Then an excess of the test item (approx. 1.11 g) was carefully added directly to the surface of the test water. The mixing vessel was thereafter 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 hours of gentle stirring in the dark and at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the saturated stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary based on the measured concentration of the stock solution (52.29 mg/L) and a fixed amount of inoculum to obtain the required test concentrations in the test vessels and a cell density of 5.103 cells/mL per vessel. The test vessels were completely filled (with minimum headspace) and were closed with ground glass stoppers. At the start of the test, test solutions in test vessels were observed to be clear and colourless at all test concentrations. The test was carried out without adjustment of the pH.
In this range-finding test, algal cells were exposed to a series of nominal test concentrations of 0.32, 1.00, 3.20, 10.0, 32.0 mg test item/L and to a concentration close to the solubility limit of the test item in test water (52.29 mg/L, slightly diluted by the algae addition  47.0 mg/L), and to a control. The test system was maintained in a temperature controlled cabinet (23°C ± 2°C) with continuous illumination for a period of 72 hours.
Based on the results of a range-finding test, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water and inoculum to obtain the following nominal concentrations (spaced by a factor of approximately 3.00): 0.30, 0.9, 2.8, 8.3 and 25.0 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.963 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1.729 - 2.180 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.919 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 3.609 - 4.276 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Remarks on result:

not determinable

Remarks:

due to mathematical reasons or inappropriate data

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1.954 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1.233 - 2.565 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.904 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 5.041 - 6.978 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Since it was not possible to determine a NOErC after 48 hours due to mathematical reasons and by taking into account the highest test concentration of 24.85 mg/L, this latter was excluded from calculations

Details on results:

- Analytical results: See Table 6.1.5/3 in "Any other information on results incl. tables". Although the geometric means of measured exposure concentrations were overall within ± 20% of the nominal concentrations, test item losses (> 20% of the initial concentrations) were observed between the start and every day until the end of the test (t=72 h). These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Moreover, analytical results showed the appearance of a new chromatographic peak from 48 hours in the highest test concentrations and at 72 hours in all test solutions, suggesting the likely formation of a degradation product. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations between the start and the end of the test (t=72h), the results were based on the geometric means of measured exposure concentrations. In addition, since analytical determinations were also performed at t=48 h and that the degradation product was most certainly less present after 48 hours than after 72 hours, the results were also based on the geometric means of measured exposure concentrations between the start of the test and t=48 h.
- Biological results: See Tables 6.1.5/1 and 2 in "Any other information on results incl. tables".
- Turbidity measurements confirmed the absence of undissolved material in the stock solution (represented by turbidity analysis of the stock solution = 0.71 FNU, in comparison with the turbidity value of the control = 0.70 FNU).

Results with reference substance (positive control):

On August 26, 2019 (KA19-002; most recent test), the 72h-EC50 was 0.719 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 to 1.73 mg/L).

Table 6.1.5/1: Algal cell densities during the final test (expressed as density of algal cells/mL x 10^4)

	Replicate	Nominal concentration and corresponding measured concentration (geometric mean) after 48 h and after 72 h (mg test item.L^-1)
		Control (0) (0)	0.30 (0.31) (0.20)	0.90 (0.82) (0.75)	2.80 (2.61) (2.27)	8.30 (8.11) (7.90)	25.00 (24.85) (24.38)
t=24 h	1	2.8	1.2	1.6	0.8	0.8	0.0
	2	2.0	2.0	1.2	1.2	1.2	0.4
	3	2.8	3.6	2.4	2.8	1.2	0.0
	4	2.0
	5	2.0
	6	1.6
	Mean	2.2	2.3	1.7	1.6	1.1	0.1
	Std. Dev.	0.49	1.22	0.61	1.06	0.23	0.23
t=48 h	1	18.0	14.4	11.2	10.0	1.6	0.4
	2	14.8	18.8	11.6	7.6	2.4	0.8
	3	13.6	17.6	12.4	9.6	1.2	0.0
	4	19.2
	5	13.2
	6	14.4
	Mean	15.5	16.9	11.7	9.1	1.7	0.4
	Std. Dev.	2.47	2.27	0.61	1.29	0.61	0.4
t=72 h	1	74.4	57.2	46.8	32.8	0.8	0.4
	2	70.8	62.4	48.0	30.0	0.8	0.4
	3	62.4	64.0	51.2	34.0	0.8	0.0
	4	72.4
	5	64.0
	6	68.8
	Mean	68.8	61.2	48.7	32.3	0.8	0.3
	Std. Dev.	4.74	3.56	2.27	2.05	0.00	0.23

Table 6.1.5/2: Mean specific growth rate in P. subcapitata during the final test

	Replicate	Nominal concentration and corresponding measured concentration (geometric mean) after 48 hand after 72 h (mg test item.L^-1)
		Control (0) (0)	0.30 (0.31) (0.20)	0.90 (0.82) (0.75)	2.80 (2.61) (2.27)	8.30 (8.11) (7.90)	25.00 (24.85) (24.38)
t=0 h - t=24 h	1	1.723	0.875	1.163	0.470	0.470	-1.609
	2	1.386	1.386	0.875	0.875	0.875	-0.223
	3	1.723	1.974	1.569	1.723	0.875	-1.609
	4	1.386
	5	1.386
	6	1.163
	Mean	1.461	1.412	1.202	1.023	0.740	-1.147
	Std. Dev.	0.2202	0.5498	0.3482	0.6392	0.2341	0.8004
	% Inh.	-	3.4	17.7	30.0	49.3	178.5
t=0 h - t=48 h	1	1.792	1.680	1.555	1.498	0.582	-0.112
	2	1.694	1.814	1.572	1.361	0.784	0.235
	3	1.652	1.781	1.605	1.477	0.438	-0.805
	4	1.824
	5	1.637
	6	1.680
	Mean	1.713	1.758	1.577	1.445	0.601	-0.227
	Std. Dev.	0.0769	0.0694	0.0259	0.0740	0.1741	0.5294
	% Inh.	-	-2.6	7.9	15.6	64.9	113.3
t=0 h - t=72 h	1	1.668	1.580	1.513	1.395	0.157	-0.074
	2	1.651	1.609	1.521	1.365	0.157	-0.074
	3	1.609	1.617	1.543	1.407	0.157	-0.536
	4	1.658
	5	1.617
	6	1.641
	Mean	1.641	1.602	1.526	1.389	0.157	-0.228
	Std. Dev.	0.0232	0.0196	0.0154	0.0215	0.0000	0.2668
	% Inh.	-	2.4	7.0	15.4	90.5	113.9

Table 6.1.5/3: Concentrations of the test item (mg/L) in test water - Final test

Nominal concentration (mg.L^-1)	Measured concentration (mg.L^-1)				Relative loss to initial value (% initial)		Geometric mean measured concentrations
	Start (t=0 h)	t=24 h	t=48 h	t=72 h
							mg.L^-1		% nominal
					(t=0 h - t=48 h)	(t=0 h - t=72 h)	(t=0 h - t=48 h)	(t=0 h - t=72 h)	(t=0 h - t=48 h)	(t=0 h - t=72 h)
Control	Abs.	Abs.	Abs.	Abs.	N.A.	N.A.	N.A.	N.A.	N.A.	N.A.
0.30	0.38	0.27	0.30	Pres.*	21	N.A.	0.31	0.20	103	67
0.90	1.00	0.73	0.76	0.57	24	43	0.82	0.75	91	83
2.80	3.14	2.41	2.35	1.49	25	53	2.61	2.27	93	81
8.30	9.65	7.37	7.51	7.30	22	24	8.11	7.90	98	95
25.00	29.56	22.98	22.59	23.03	24	22	24.85	24.38	99	98

N.A.: not applicable

% = Percent of expected nominal concentration in test item.

*Pres= Presence: concentrations below the LOQ (0.10 mg/L) but above the LOD (0.03 mg/L). Where measured concentrations were below the LOQ but above the LOD, such concentrations were considered to be the half of the quantification limit (LOQ/2 = 0.05 mg/L).

Abs= Absence: concentrations below the LOQ (0.10 mg/L) and the LOD (0.03 mg/L).

Validity criteria fulfilled:

yes

Remarks:

See "Overall remarks"

Conclusions:

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test. Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72h-ErC50 and 72h-ErC10 values for growth rate were determined at 3.919 mg/L and 1.963 mg/L, respectively.

Executive summary:

This study was performed according to OECD Guideline 201 and EU Method C.3 with GLP compliance to assess the test substance for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata.

Following a preliminary range-finding test, algal cells were exposed to an aqueous solution of the test substance over 72 hours at the required nominal test concentrations 0.30, 0.9, 2.8, 8.3 and 25.0 mg/L, under closed and static conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Samples taken from the control and all test concentrationswere analysed at the start and every day thereafter until the end of the test in order to determine if concentrations of the test item were maintained.

Specific analyses of samples taken from all test concentrations revealed test item losses (>20% of the initial concentrations) between the start and every day until the end of the test. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were based on the geometric means of measured exposure concentrations.

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72h-ErC50 and 72h-ErC10 values for growth rate were determined at 3.919 mg/L and 1.963 mg/L, respectively.

Since the 72h-NOECr value for growth rate could not be determined, the endpoint values at t=48h were also determined in order to have a complete set NOEC/EC10/EC50.

Indeed, according to OECD Guideline 201 section 11, it is possible to shorten the test period to 48 hours whenever unlimited, exponential growth during the test has been obtained. This corresponds to at least a 16-fold growth in controls. Since the cell density in the control cultures increased by a factor of 31 within 48 hours exposure period, the results can also be provided after 48 h (other validity criteria of the study at t=48 h were also respected). Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 48h-ErC50 and 48h-ErC10 values for growth rate were determined at 5.904 mg/L and 1.954 mg/L, respectively. The 48h-NOECr value for growth rate was 0.310 mg/L.

Description of key information

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

72h-ErC50 (Pseudokirchneriella subcapitata) = 3.919 mg/L (95% CL: 3.609 - 4.276 mg/L) based on geometric mean measured concentrations.

72h-ErC10 (Pseudokirchneriella subcapitata) = 1.963 mg/L (95% CL: 1.729 - 2.180 mg/L) based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.919 mg/L

EC10 or NOEC for freshwater algae:

1.963 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 an aqueous solutionof the test substance over 72 hours at the required nominal test concentrations 0.30, 0.9, 2.8, 8.3 and 25.0 mg/L, under closed and static conditions. Specific analyses of samples taken from all test concentrations revealed test item losses (>20% of the initial concentrations) between the start and every day until the end of the test. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were based on the geometric means of measured exposure concentrations. Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72h-ErC50 and 72h-ErC10 values for growth rate were determined at 3.919 mg/L (95% CL: 3.609 - 4.276 mg/L) and 1.963 mg/L (95% CL: 1.729 - 2.180 mg/L), respectively.