[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate

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:

2017-06-12 to 2017-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 201. The validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on January 10, 2017

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test concentrations at the start and the end of the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The mixing vessel was a cylindrical glass bottle sealed with a screw cap. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and 1 L test water was added. Then 105.20 mg test item (gelled texture) was weighed on a glass slide that afterwards was placed under the surface of the test water contained in the mixing vessel through fishing wire. 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 24 ± 2 hours of gentle stirring in the dark and at approx. 40°C, the contents of the vessel was allowed to stand undisturbed for ca. 5 min at room temperature. Then the stock solution was diluted with test water as necessary and a fixed amount of inoculum to obtain the required test concentrations in the test vessels and a cell density of 5.10^3 cells.mL-1 per vessel. The test was carried out without adjustment of the pH. At the start of the test, the solution was observed to be clear and colourless.

- Controls: Test water without test substance but treated in the same way as the test substance solutions (WAFs).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): 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: 2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1.10^4 cells.mL-1. 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

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

No data

Test temperature:

22.8-22.9 °C throughout the test (average value: 22.9 °C)
This complied with the requirements (23°C ± 2°C, constant within 2°C).

pH:

Start(t=0 h): 8.29 - 8.50
End (t=72 h): 8.33 - 10.15
Although the pH level in the control varied more than 1.5 units at the end of the test (1.86 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the addition of more sodium bicarbonate.

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

no data

Nominal and measured concentrations:

Nominal concentrations: 0.8; 2.8; 8.0; 25.3 and 80.0 mg/L
Corresponding measured concentrations: 0.52; 4.60; 7.29; 24.82 and 79.11 mg/L (geometric means of measured exposure concentrations 0 - 72h)

Details on test conditions:

TEST CONDITIONS
- Test vessel: 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.
- Initial cells density: An initial cell density of 5.10^3 cells.mL-1 using the exponentially growing pre-culture.
- Replicates: 6 controls and 3 replicates of each test concentration for counting. Moreover, a replicate of each treatment with algae was prepared for chemical analyses.
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.

GROWTH MEDIUM
- Standard medium used: Yes; Original medium of OECD TG 201. Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- 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:
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- Other:
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=0 h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

RANGE-FINDING TEST:
In the range-finding test, algal cells were exposed to a series of nominal test concentrations of 1.0, 10.0, 20.0 and 100.0 mg test item.L-1. The test conditions were the same than the final test. The % inhibition reported at 1.0; 10.0; 20.0 and 100.0 mg/L respectively were:
- 7.0; 24.5; 84.9 and 66.3 at t=24h
- 3.6; 13.7; 56.5 and 102.2 at t=48h
- 14.4; 27.0; 63.0 and 108.4 at t=72h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Results used to determine the conditions for the definitive study: Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following concentrations: 0.8, 2.5, 8.0, 25.3 and 80.0 mg.L-1.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.733 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 5.182 - 6.265 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.938 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: 2.195 - 3.481 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 7.591 - 8.789 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.87 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: 3.195 - 4.346 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.265 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 15.423 - 17.163 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.553 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: 6.104 - 7.132 mg/L

Results with reference substance (positive control):

On February 14, 2017 (most recent test), the 72h-EC50 was 1.438 mg.L-1 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-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

Reported statistics and error estimates:

The software ToxRat® Professional was used to perform statistical analyses. No reported statistics and error estimates.

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

	Nominal concentration(mg test item.L^-1)
	Control	0.8	2.5	8.0	25.3	80.0
Start t=0h	8.29	8.36	8.39	8.38	8.42	8.50
End t=72h	10.15	10.05	10.00	9.75	8.77	8.33

Table 6.1.5/2: Algal cell densities during the final test (expressed as density of algal cells/mL x 10⁴)

	Replicate	Nominal concentration(mg test item.L^-1) and correspondingmeasured concentration(mg test item.L^-1) (geometric means ofmeasured exposure concentrations0 - 72h)
		Control (0)	0.8 (0.52)	2.8 (4.60)	8.0 (7.29)	25.3 (24.82)	80.0 (79.11)
t=24h	1	4.8	4.4	4.8	3.2	2.0	0.8
	2	4.8	4.8	4.8	2.8	1.6	0.4
	3	6.0	4.8	6.0	4.0	2.0	0.4
	4	5.2
	5	5.2
	6	4.8
	Mean	5.1	4.7	5.2	3.3	1.9	0.5
	Std. Dev.	0.47	0.23	0.69	0.61	0.23	0.23
t=48h	1	29.2	28.4	29.2	15.6	2.8	0.8
	2	28.0	27.6	26.8	12.8	2.4	0.8
	3	27.6	27.6	30.4	14.4	2.0	0.4
	4	30.4
	5	28.4
	6	28.4
	Mean	28.7	27.9	28.8	14.3	2.4	0.7
	Std. Dev.	1.00	0.46	1.83	1.40	0.40	0.23
t=72h	1	84.0	91.2	60.4	38.0	2.4	0
	2	111.2	86.8	67.2	41.6	2.4	0.8
	3	85.6	84.0	74.0	42.4	2.4	0
	4	94.4
	5	88.4
	6	98.8
	Mean	93.7	87.3	67.2	40.7	2.4	0.3
	Std. Dev.	10.20	3.63	6.80	2.34	0.00	0.40

 

At test start 5000 algal cells.mL^-1 were incubated; 6 replicates of the controls and 3 replicates of each test concentration.

Std. Dev.: standard deviation.

 

Validity criteria of the study

- Cell density in Controls: 187.5-fold increase within 72 hours. The specific growth rate was determined at 1.74 day^-1, so greater than 0.92 day^-1.

- Coefficient of Variation:

1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 33.2%.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 2.0%.

Thus the validity criteria were respected in this study.

 

Analytical results

Chemical analyses revealed slight test item losses, especially at the lowest test concentrations, and a relative stability at the highest test concentrations (where toxicity was more pronounced, see table below):

 

Nominal concentration (mg test item.L^-1)	Start (t=0h)	End (t=72h)	Relative loss to initial value (t=0h - t=72h) (%)	Geometric mean measured concentration
				mg.L^-1	%
Control	Absence	Absence	N.A.	N.A.	N.A.
0.8	Presence	Presence	N.A.	0.52*	N.A.
2.5	5.74	3.69	36	4.60	184
8.0	8.79	6.05	31	7.29	91
25.3	25.64	24.02	6	24.82	98
80.0	80.16	78.07	3	79.11	99

N.A.: not applicable

Absence: concentrations below the LOQ (1.04 mg.L^-1) and the LOD (0.31 mg.L^-1).

Presence: concentrations below the LOQ (1.04 mg.L^-1) and above the LOD (0.31 mg.L^-).

% = Percent of expected nominal concentration in test item

Samples were centrifuged before analysis.

*Where measured concentrations were below the LOQ, such concentrations were considered to be half the quantification limit, according to the study plan.

This could suggest metabolism and/or adsorption effects of the test item by P. subcapitata. 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 ofmeasured exposure concentrations.

 

Nominal concentration (mg test item.L^-1)	0.80	2.50	8.0	25.3	80.0
Correspondingaverage exposure concentrations (geometric means) (mg test item.L^-1)	0.52	4.60	7.29	24.82	79.11

 

Physico-chemical parameter values throughout the test

Although the pH level in the control varied more than 1.5 units at the end of the test (1.86 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the addition of moresodium bicarbonate.

The mean light intensity was of 5346 lux (range: 4544-6148 lux), which remained within the ranges prescribed by the study plan (range: 4440-8880 lux and shall not vary more than ± 15% from the average light intensity over the incubation area).

The temperature in the incubator was situated between 22.8 and 22.9°C throughout the test (average value: 22.9°C), and complied with the requirements (23°C ± 2°C, constant within 2°C).

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 16.265 mg.L-1 and 6.553 mg.L-1, respectively. The 72-hour EC10 for growth rate was 5.733 mg L-1 and for yield was 2.938 mg.L-1.

Executive summary:

A study was performed to assess the test item QUESTICE L 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 for Testing of Chemicals No. 201 and was performed under GLP compliance.

Following a preliminary range-finding test, algal cells were exposed to an aqueous solutionof the test item over a range of nominaltest concentrationsof 0.8, 2.5, 8.0, 25.3and 80.0mg/L. 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 were determined by chemical analysesat the start and the end of the test.

Chemical analyses revealed slight test item losses, especially at the lowest test concentrations, and a relative stability at the highest test concentrations (where toxicity was more pronounced). This could suggest metabolism and/or adsorption effects of the test item byP. subcapitata. 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

After 72 hours of exposure, theE_rC_xand E_yC_xwere as follows:

Parameter	Growth rate (mg.L^-1)	Yield (mg.L^-1)
72-hour EC10	5.733	2.938
95% conf. limits	5.182 – 6.265	2.195 – 3.481
72-hour EC20	8.200	3.870
95% conf. limits	7.591 – 8.789	3.195 – 4.346
72-hour EC50	16.265	6.553
95% conf. limits	15.423 – 17.163	6.104 – 7.132

Statistical analyses were performed by the computer program ToxRat.

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 16.265 mg.L-1 and 6.553 mg.L-1, respectively. The 72-hour EC10 for growth rate was 5.733 mg L-1 and for yield was 2.938 mg/L.

Description of key information

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72-hour ErC50 for the parameters growth rate was determined to be 16.265 mg/L. The 72-hour ErC10 was 5.733 mg/L (growth rate).

Key value for chemical safety assessment

EC50 for freshwater algae:

16.265 mg/L

EC10 or NOEC for freshwater algae:

5.733 mg/L

Additional information

A reliable experimental study was available to assess the ability of the registered substance QUESTICE L to generate toxic effects on Pseudokirchneriella subcapitata, under static conditions. This study was performed under GLP compliance and according to the OECD 201 guideline.

Following a preliminary range-finding test, algal cells were exposed to an aqueous solutionof the test item over a range of nominaltest concentrationsof 0.8, 2.5, 8.0, 25.3and 80.0mg/L. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.

Chemical analyses revealed slight test item losses greater than ± 20% of the initial concentrations (especially at the lowest test concentrations) and therefore the results were based on the geometric means of measured exposure concentrations.

Besides some pH values that did not meet the requirements (but deviation not affecting the integrity of the study), all the validity criteria of the study as laid down in the guideline were respected. Therefore, this study was considered acceptable for that endpoint.