13-ethyl-11-methylene-gon-4-ene-3,17-dione

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 July 2020 - 03 Sep-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD series on testing and assessment number 23 (2nd edition),

Version / remarks:

2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione

Cas Number:

54024-17-8

Molecular formula:

C20 H26 O2

IUPAC Name:

(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione

Test material form:

solid: particulate/powder

Details on test material:

Storage Conditions: In refrigerator (2-8°C)

Specific details on test material used for the study:

Physical Description: Off-white powder
Storage conditions: In refrigerator (2-8°C)
Test item handling: No specific handling conditions required

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
- Frequency: at t=0 h and t=72 h.
- Volume: 2.0 mL from the approximate centre of the test solutions.
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test solution containing 32% of the SS, but without algae and samples for analysis were taken at the start and at the end of the test period.
- Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible
analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum
of three months after delivery of the draft report, pending on the decision of the sponsor for
additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10E^4 cells/mL.

Test organisms

Test organisms (species):

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

Details on test organisms:

Test System
Species: Raphidocelis subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.

Breeding:
- Stock culture: Algae stock cultures were started by inoculating growth medium 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 21-24°C.
- Light intensity 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

- Pre-culture: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

between 22 and 24°C.

pH:

Start of the test: 8.0 - 8.1
End of the test: 7.8 - 8.0

Nominal and measured concentrations:

Nominal concentration: Solutions containing 10, 18, 32, 56 and 100% of the SS,
prepared at a loading rate of 100 mg/L.
Mean Measured concentrations: the average exposure concentration was 3.0, 5.6, 10, 18 and 30 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessels: 100 mL, all-glass with aluminum caps, perforated for ventilation, containing 50 mL of test solution
- Test Medium: M2
- Cell density: An initial cell density of 1 x 10^4 cells/mL
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Replicates: 3 replicates of each test concentration, 6 replicates of the control, 1 replicate of each test concentration without algae.

GROWTH MEDIUM
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis) added with analytical grade salts.
- Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water

TEST MEDIUM
- Test medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water, added with analytical grade salts.

OTHER TEST CONDITIONS
- Sterile test conditions: no, aseptic conditions
- Adjustment of pH: no
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 91 to 94 μE.m-2.s-1.

EFFECT PARAMETERS MEASURED :
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter (24, 48 and 72h), cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank.
At the end of the final test microscopic observations were performed on the test vessel containing 32% of the SS to observe for any abnormal appearance of the algae compared to the control.

RANGE-FINDING:
Three replicates of exponentially growing algal cultures were exposed to solutions containing 0.10, 1.0, 10, and 100% of a SS prepared at a loading rate of 100 mg/L.
No growth rate and yield inhibition was recorded at the three lowest concentrations tested,
while growth rate and yield were inhibited by 57 and 93% at the highest test concentration,
respectively. These results were used to determine the concentrations of the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate The batch of Raphidocelis subcapitata tested (September 2020) , showed expected sensitivity to Potassium dichromate based on the historical range of reference tests performed by the Test Facility in the last ten years

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 10-12 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72

Dose descriptor:

EC50

Effect conc.:

33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 31-35 mg/L

Details on results:

Analytical results: For detail see Table analytical results in "any other information on results".
Samples taken from all test concentrations and the control were analysed. The measured
concentrations at the start of the test were 3.2, 5.8, 11, 18, and 31 mg/L in solutions containing
10, 18, 32, 56, and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. The
concentrations remained stable during the exposure period, i.e. were at 89 - 99% relative to
the initial values at the end of the test. The average exposure concentration were 3.0, 5.6, 10, 18, and 30 mg/L.
The concentrations measured in the samples taken from solution without algae (abiotic
control) were comparable with the concentrations measured in the samples with algae.
A small test item response was detected in the sample taken from the control at the end of the test. The origin of this response is unknown. Since this response is negligible compared to the lowest test concentration (maximum contribution is 0.00079%), it was considered that this had no impact on the condition of the algae in the control group.


Biological results: For details see tables in "any other information on results".
Inhibition of growth rate and yield increased with increasing concentration of EMETAM
from 5.6 mg/L upwards, resulting in 44% growth rate inhibition and 92% yield inhibition at
the highest concentration tested. Statistically significant inhibition of growth rates was found
at all test concentrations. Hence, the NOEC could not be determined based on the results of
this experiment. Nevertheless, the 72h-EC10 can be used as a surrogate parameter for the
NOEC.
Microscopic observations at the end of the test revealed a normal and healthy appearance of
the algal cells exposed to an average exposure concentration of 10 mg/L when compared to
the control.

Validity of the Test
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 294).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 27%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 1.0%).

Reported statistics and error estimates:

For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on Weibull analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the
analysis.

Any other information on results incl. tables

Table: Measured test item concentration and average exposure concentration

EMETAM Concentration (%SS  of 100 mg/L)	t=0h (mg/L)	T=72 h (mg/l)	Average exposure concentration (mg/L)
10	3.2	2.9	3.0
18	5.8	5.3	5.6
32	11	9.4	10
56	18	18	18
100	31	30	30

 

Table:  Individual cell densities (x10^4)

Time	Replicate	Control	3.0 mg/L	5.6 mg/L	10 mg/L	18 mg/L	30 mg/L
0h	1	1.0	1.0	1.0	1.0	1.0	1.0
	2	1.0	1.0	1.0	1.0	1.0	1.0
	3	1.0	1.0	1.0	1.0	1.0	1.0
	4	1.0
	5	1.0
	6	1.0
24h	1	11.374	11.422	11.113	9.716	8.210	5.515
	2	12.097	10.486	10.038	9.411	8.188	5.415
	3	11.831	11.013	11.383	10.273	8.980	6.064
	4	11.666
	5	12.075
	6	10.995	65.337	67.331	49.545	33.348	16.846
48h	1	66.399	61.319	56.814	48.948	32.151	14.595
	2	71.762	62.691	63.000	47.595	34.601	15.496
	3	68.802
	4	68.967
	5	72.680
	6	64.584
72	1	293.146	269.245	271.621	199.324	89.065	24.694
	2	296.728	242.505	243.980	181.285	79.536	23.358
	3	290.539	251.210	258.593	183.662	92.621	23.401
	4	315.724
	5	302.902
	6	264.966

 

Table: Growth rate an percenatge inhibition for the total test period

EMETAM Average concentration (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	1.9	0.019	6
3.0	1.8	0.018	3	2.5*
5.6	1.9	0.018	3	2.3*
10	1.7	0.017	3	7.9*
18	1.5	0.026	3	21*
30	1.1	0.011	3	44*

*Effect was statistically significant

Table: Effect parameters

Parameter  (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	<3.0	11	17	33
	lower 95% CI		10	16	31
	upper 95% CI		12	18	35
Yield	Value	<3.0	4.0	6.4	13
	lower 95% CI		3.0	5.3	12
	upper 95% CI		4.9	7.4	14

CI: Confidence interval

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

for details see" detail on results"

Conclusions:

The 72h-EC50 for growth rate inhibition (ERC50) was 33 mg/L with a 95% confidence interval
ranging from 31 to 35 mg/L. The 72h-EC10 for growth rate inhibition (ERC10) was 11 mg/L with a 95% confidence interval ranging from 10 to 12 mg/L.

The 72h-NOEC for growth rate was lower than 3.0 mg/L.

Executive summary:

Toxicity to aquatic algae Raphidocelis subcapitata was assessed according to OECD guideline 201 and in compliance with GLP. Algae were exposed under static conditions to the test substance at 10, 18, 32, 56 and 100% of the saturated solution prepared at a loading rate of 100 mg/L in fresh water. The test was performed with 3 replicates per concentration and 6 replicates for the control.The measured concentrations at the start of the test were 3.2, 5.8, 11, 18 and 31 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS, respectively. The concentrations remained stable during the exposure period, i.e. were at 89 - 99% relative to the initial values at the end of the test. The average exposure concencentration was calculated to be 3.0, 5.6, 10, 18, and 30 mg/L.

Inhibition of growth rate and yield increased with increasing concentration of EMETAM from 5.6 mg/L upwards, resulting in 44% growth rate inhibition and 92% yield inhibition at the highest concentration tested. Statistically significant inhibition of growth rates was found at all test concentrations. Hence, the NOEC could not be determined based on the results of this experiment.

The 72h-EC50 for growth rate inhibition (ERC50) was 33 mg/L with a 95% confidence interval ranging from 31 to 35 mg/L. The 72h-EC10 for growth rate inhibition (ERC10) was 11 mg/L with a 95% confidence interval ranging from 10 to 12 mg/L. The 72h-NOEC for growth rate inhibition was lower than 3.0 mg/L.

All the validity criteria were met, therefore the study was considdered to be valid.