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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:

22 Jul - 14 Sep 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006, corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

Cas Number:

18641-58-2

Molecular formula:

C69H132O8

IUPAC Name:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

Sampling and analysis

Analytical monitoring:

yes

Remarks:

UPLC-MS/MS

Details on sampling:

- Concentrations: Samples were taken after 0h, 24h and 72h from all test concentrations (i.e. test vessels at 1.25, 2.5, 5.0, 10 and 20 μg/L inoculated with algae), from the abiotic control (i.e. test vessel at 5.0 μg/L without algae) and from the blank and the solvent controls. Stock solutions used for the preparation of test solutions were sampled as well. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (set to maintain -20°C) until analysis at the analytical laboratory of the Test Facility.

Test solutions

Vehicle:

yes

Remarks:

tetrahydrofuran (THF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Preliminary testing: A non-GLP saturation test was performed in order to select the most appropriate preparation procedure for test solutions. A loading rate of 100 mg/L was continuously stirred for 3 days and concentrations were measured every 24 hours after filtration using a 0.45 μm syringe filter. The measured concentrations in the samples taken after 24, 48 and 72 hours of magnetic stirring were 0.42, 0.63 and 0.33 μg/L by extrapolation of the calibration curve (i.e. below LOQ = 1 μg/L of analytical method in preliminary testing). Based on the obtained results, it was concluded that preparation of test solutions using solvent stocks was required to enable reliable quantification of the test material during the final test.
- Method: The test item was poorly water soluble under test conditions, as confirmed in preliminary testing. Therefore, to enable reliable preparation of test concentrations and reliable quantification of the test material, the test solutions were prepared using a suitable organic solvent according to the OECD Guidance Document 23. The preparation of the stock solutions for the final test, i.e. test material in tetrahydrofuran (THF), started with the highest concentration of 0.20 mg/mL. The highest concentrated stock solution was vigorously shaken to completely dissolve the test material in THF. The lower stock concentrations, i.e. 0.0125, 0.025, 0.050 and 0.10 mg/mL, were prepared by subsequent dilutions of the highest concentrated stock in THF. Subsequently, a volume of 100 μL of each stock solution was added to respectively 1.0 L test medium to obtain the according test concentrations. The mixtures were magnetically stirred for 15 minutes.
- Controls: Blank control (test medium only) and solvent control (prepared by adding 100 μL THF to 1.0 L test medium)
- Other controls: Abiotic control (i.e. test vessel at 5.0 μg/L without algae) to check maintenance of actual concentrations
- Chemical name of vehicle: Tetrahydrofuran (THF)
- Concentration of vehicle in test medium: 100 μL/L in all test concentrations and in solvent control. Organic solvent concentrations up to 100 μL/L are allowed according to OECD 201.
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: The algae were cultured in OECD medium at a temperature of 21-24°C and continuously aerated and exposed to light (light intensity 60 to 120 μE/m2/s in wavelength range of 400 to 700 nm).
- Age of inoculum: A pre-culture was prepared from the algal stock culture 3 days before the start of the test under the same conditions as used in the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22 - 23°C

pH:

8.0 - 8.2

Nominal and measured concentrations:

Nominal: Controls (blank and solvent), 1.25, 2.5, 5.0, 5.0 (abiotic control without algae), 10 and 20 μg/L
Measured (0h): < LOQ, 1.20, 2.29, 4.90, 5.05, 9.67 and 17.8 μg/L
Measured (24h): < LOQ, < LOQ, < LOQ, < LOQ, < LOQ, 1.10 and 0.876 μg/L
Measured (72h): < LOQ for all controls and all test concentrations
Measured (TWA): < LOQ, 0.21, 0.26, 0.34, 0.35, 1.3 and 1.5 μg/L

LOQ = 0.25 μg/L in the final test

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminum caps, perforated for ventilation, containing 50 mL of test solution.
- Initial cells density: 1x10^4 cells/mL
- Control end cells density: 218x10^4 cells/mL in the blank control and 233x10^4 cells/mL in the solvent control
- No. of vessels per concentration: 3 replicates. Extra replicates (1-2) per concentration were prepared without algae for sampling/effect parameters measurements
- No. of vessels per blank control: 6 replicates
- No. of vessels per vehicle control: 6 replicates

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium M2
- Culture medium different from test medium: OECD medium M1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity: 79 to 80 μE/m2/s
- Other: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 20 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes, a non-GLP range-finding test was performed at a loading rate of 100 mg/L. The test solution was stirred with a magnetic stirrer for 3 days and then the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 μm membrane filter and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. No inhibition of algal growth rate or yield was found at any of the tested concentrations. An additional GLP range finding test was conducted using 0.25, 1.0 and 4.0 μg/L test concentrations (prepared using THF similarly as in the final test), a blank control and a solvent control. No significant inhibition of algal growth rate or yield was found at any of the test groups in the additional range-finding test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.5 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1.5 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.5 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes (dating March 2022)
- EC50 (72 h): 0.95 mg/L, 95% confidence limits: 0.87 - 1.0 mg/L

Reported statistics and error estimates:

ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used to perform the statistical analysis. 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 (Dunnett`s Multiple t-test Procedure, α=0.05, one-sided, smaller). This statistical comparison was preceded by a check on normal distribution of the data (Shapiro-Wilk’s Test) and a test for homogeneity of the variances (Levene’s test).
The ECx-values could not be determined because the observed effects were below 10%.

Any other information on results incl. tables

VALIDITY CRITERIA

The study fulfilled the validity criteria defined by the guideline (Table 1) and is thus considered valid and reliable.

Table 1: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	Actual values: 218 in the blank control and 233 in the solvent control	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	Actual values: 26% in the blank control and 10% in the solvent control	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata.	Actual values: 1.3% in the blank control and 2.2% in the solvent control	Yes

ANALYTICAL RESULTS

The analytical findings are summarized in Table 2.

Table 2. Analytical results

Nominal concentration (μg/L)	Measured concentration (μg/L) at 0h	Measured concentration (μg/L) at 24h	Measured concentration (μg/L) at 72h	TWA (μg/L)
Controls (blank and solvent)	<LOQ	<LOQ	<LOQ	<LOQ
1.25	1.20	<LOQ*	<LOQ*	0.21
2.5	2.29	<LOQ*	<LOQ*	0.26
5.0	4.90	<LOQ*	<LOQ*	0.34
5.0**	5.05	<LOQ*	<LOQ*	0.35
10	9.67	1.10	<LOQ*	1.3
20	17.8	0.876	<LOQ*	1.5

*Limit of Quantification (LOQ) = 0.25 μg/L. Therefore, half of the LOQ (i.e. 0.125 μg/L) was used to calculate the time-weighted average exposure concentrations (TWA).

**Abiotic control (without algae)

 

BIOLOGICAL RESULTS
Biological findings are summarised in Table 3, Table 4, Table 5 and Table 6.

Table 3. Results in control replicates

	Individual Cell Densities [x10^4cells/mL]				Section-by-Section Growth Rates [day^-1]			Growth rates [day^-1]	CV growth [%]	Yields [x10^4cells/mL]	CV yield [%]
	0 h	24 h	48 h	72 h	0 – 24 h	24 – 48 h	48 – 72 h	72 h	72 h	72 h	72 h
Blank Control	1.000	3.233	39.570	201.410	1.173	2.505	1.627	1.768	1.3	200.410	7.1
	1.000	3.875	39.147	202.334	1.355	2.313	1.643	1.770		201.334
	1.000	4.963	41.661	218.730	1.602	2.128	1.658	1.796		217.730
	1.000	4.944	46.388	211.550	1.598	2.239	1.517	1.785		210.550
	1.000	4.197	44.675	237.434	1.434	2.365	1.670	1.823		236.434
	1.000	4.349	43.755	233.746	1.470	2.309	1.676	1.818		232.746
Mean	1.000	4.260	42.533	217.534	1.439	2.310	1.632	1.793		216.534
Solvent Control	1.000	6.671	40.228	214.998	1.898	1.797	1.676	1.790	2.2	213.998	12.4
	1.000	6.702	37.779	196.209	1.902	1.729	1.647	1.760		195.209
	1.000	7.666	47.821	251.649	2.037	1.831	1.661	1.843		250.649
	1.000	8.690	44.859	230.742	2.162	1.641	1.638	1.814		229.742
	1.000	6.993	40.191	225.212	1.945	1.749	1.723	1.806		224.212
	1.000	8.723	51.890	278.278	2.166	1.783	1.680	1.876		277.278
Mean	1.000	7.574	43.795	232.848	2.018	1.755	1.671	1.815		231.848

Table 4. Results in treatments.

Nominal concentration [μg/L]	Cell Densities [x10^4cells/mL]				Growth rates [day^-1]	CV growth [%]	Yields [x10^4cells/mL]	CV yield [%]
	0 h	24 h	48 h	72 h	72 h	72 h	72 h	72 h
1.25	1.000	7.378	44.159	213.352	1.788	1.1	212.352	6.2
	1.000	7.308	42.711	225.828	1.807		224.828
	1.000	9.243	46.738	241.237	1.829		240.237
Mean	1.000	7.976	44.536	226.806	1.808		225.806
2.5	1.000	6.449	37.240	199.363	1.765	2.0	198.363	11.2
	1.000	7.166	39.551	197.409	1.762		196.409
	1.000	9.068	43.486	239.339	1.826		238.339
Mean	1.000	7.561	40.092	212.037	1.784		211.037
5.0	1.000	6.984	40.408	221.013	1.799	1.9	220.013	10.1
	1.000	6.940	36.685	197.248	1.761		196.248
	1.000	9.029	48.391	241.314	1.829		240.314
Mean	1.000	7.651	41.828	219.858	1.797		218.858
10	1.000	5.901	33.713	183.285	1.737	2.9	182.285	14.9
	1.000	6.835	40.830	226.365	1.807		225.365
	1.000	7.547	46.367	247.137	1.837		246.137
Mean	1.000	6.761	40.303	218.929	1.794		217.929
20	1.000	6.657	38.038	208.280	1.780	0.7	207.280	3.8
	1.000	6.937	42.716	214.148	1.789		213.148
	1.000	7.178	44.906	224.248	1.804		223.248
Mean	1.000	6.924	41.887	215.559	1.791		214.559

Table 5. Yields/Growth rates and Percentage Inhibition for the Total Test Period

Nominal concentration [μg/L]	Growth rate (mean) [day^-1]	Growth rate inhibition [%] compared to control	Yield (mean) [x10^4cells/mL]	Yield inhibition [%] compared to control
	72 h	72 h	72 h	72 h
Solvent control	1.815		231.8
1.25	1.808	0.39	225.8	2.6
2.5	1.784	1.7	211.0	9.0
5.0	1.797	1.0	218.9	5.6
10	1.794	1.2	217.9	6.0
20	1.791	1.3	214.6	7.5

Table 6. Effect values based on Initial Measured Concentrations and Average Exposure Concentrations (TWA)

Effect parameters	Initial Measured Concentrations	Average Exposure Concentrations (TWA)
NOEC (growth and yield)	≥ 20 μg/L	≥ 1.5 μg/L
ErC10 and EyC10 (72 h)	> 20 μg/L	> 1.5 μg/L
ErC50 and EyC50 (72 h)	> 20 μg/L	> 1.5 μg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.