Adenine

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:

2018-09-17 to 2018-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of each concentration and analyzed. At the end of the exposure (72 hours), samples of the old media were taken from the test vessels for analyses.
- Sample storage conditions before analysis: If necessary, all original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal concentration of 100 mg/L of the test item was freshly prepared prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of dilution water into the glass flask. This dispersion was treated with ultrasound for 30 minutes at room temperature. Care was taken to avoid bacterial contamination. The test solutions were prepared by diluting the stock solution with dilution water.
- Controls: Six replicates (without test item) were tested under the same test conditions as the test item replicates. Potassium dichromate was tested twice per year as a reference item.
- Evidence of undissolved material: No

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: HINDAK, CCAP 278/4
- Synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd
Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2590 - 5180 lux per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Nominal hardness of medium: 0.24 mmol Ca+Mg/L.

Test temperature:

21 - 24°C ± 2°C

pH:

7.74 - 9.31

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal: 0.768,1.92, 4.80, 12.0, 30.0 mg/L
Mean measured: 2.06, 4.71, 12.1, 28.9 mg/L (fresh media, 0 hours); 1.68, 4.84, 12.0, 29.9 mg/L (old media, 72 hours)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: Glass, nominal volume: 250 mL, fill volume: 100 mL, sealed with cotton wool plugs
- Aeration: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: 6442 cells/mL
- Control end cells density: 2084121 cells/mL (mean)
- No. of vessels per concentration: 3
- No. of vessels per blank control: 6

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultra-pure water used for preparation of test medium

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuously illuminated
- Light intensity: 2590 - 5180 lux
- Light homogeneity (target): Within ± 15% over incubation area

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Chlorophyll a-fluorescence

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: Non-GLP preliminary test performed
- Test concentrations: 1.00, 10.0, 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 3.57 - 4.56 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 2.20 - 3.70

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 2.58 - 3.92 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 2.23-6.65 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 2.23-6.65 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence intervals: 2.11 -6.45 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 2.02 - 6.40 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities: No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 1.09 mg/L (growth rate), 0.548 mg/L (yield)

Reported statistics and error estimates:

Shapiro-Wilk's test on normal distribution was calculated with a significance level of 0.01.
Levene's test on variance homogeneity was calculated with a significance level of 0.01.
Monotonicity of Concentration/Response was calculated by Trend Analysis by Contrasts (significance level 0.05) for yield.
Monotonicity of Concentration/Response was calculated by non-parametric trend analysis by contrasts (significance level 0.05) for growth rate.
SD Jonckheere-Terpsta test procedure was done with a significance level 0.05.

Table 1: Cell Densities

Nominal test item concentration[mg/L]	Replicate	Cell density [cells/mL]
	No.	0 hours	24 hours	48 hours	72 hours
30	1	6442	8224	9376	7509
	2	6442	9811	10409	7284
	3	6442	7842	10824	7909
	Mean	6442	8626	10203	7567
12	1	6442	11573	14426	11501
	2	6442	9495	14262	11258
	3	6442	7256	10992	11501
	Mean	6442	9441	13227	11420
4.8	1	6442	15270	33819	48545
	2	6442	16799	42724	75540
	3	6442	13686	30547	49670
	Mean	6442	15252	35697	57918
1.92	1	6442	25880	244618	2035762
	2	6442	20220	200966	2030648
	3	6442	26401	190162	2017242
	Mean	6442	24167	211915	2027884
0.768	1	6442	27481	221451	2003986
	2	6442	23591	259295	1909998
	3	6442	25676	256564	2084617
	Mean	6442	25583	245770	1999534
Control	1	6442	24224	201641	2118181
	2	6442	30912	249365	1772964
	3	6442	27652	288737	2046436
	4	6442	27834	300832	2057061
	5	6442	24388	292927	2132877
	6	6442	20250	209565	2377205
	Mean	6442	25877	257178	2084121

Table 2: Evaluation after 72 hours. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item concentration [mg/L]	Replicate No.	Growth Rate [d^-1]	Inhibition of Growth Rate [%]	Yield [cells/mL]	Inhibition of Yield [%]
30.0	1	0.051	97	1067	100
	2	0.041	98	842	100
	3	0.068	96	1467	100
	Mean	(+) 0.053	97	1125	100
12.0	1	0.193	90	5059	100
	2	0.186	90	4816	100
	3	0.193	90	5059	100
	Mean	(+) 0.191	90	(+) 4978	100
4.80	1	0.67	65	42103	98
	2	0.82	57	69098	97
	3	0.68	65	43228	98
	Mean	(+) 0.73	62	(+) 51476	98
1.92	1	1.92	0	2029320	2
	2	1.92	0	2024206	3
	3	1.92	1	2010800	3
	Mean	(-) 1.92	0	(-) 2021442	3
0.768	1	1.91	1	1997544	4
	2	1.9	1	1903556	8
	3	1.93	0	2078175	0
	Mean	(-) 1.91	1	(-)1993992	4
Control	1	1.93		2111739
	2	1.87		1766522
	3	1.92		2039994
	4	1.92		2050619
	5	1.93		2126435
	6	1.97		2370763
	Mean	1.93		2077679

Table 3: Measured Concentration of the test item in the Definitive Test with Algae

Sampling date	Fresh Media, 0 hours		Old Media, 72 hours
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	%	Meas. conce. [mg/L]	%
30	28.9	96	29.9	100
12	12.1	101	12	100
4.8	4.71	98	4.84	101
1.92	2.06	107	1.68 *	88
0.768	< LOQ		< LOQ
Control	< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percentage of the nominal measured concentration of the test item

LOQ = limit of quantification (1.80 mg/L of the test item)

*) = > 70% LOQ

Validity criteria fulfilled:

yes

Conclusions:

In a growth inhibition test with Raphidocelis subcapitata according to OECD TG 201, EU Method C.3 and GLP principles, the 72-hour ErC50 was 4.33 mg/L and the EyC50 was 3.93 mg/L (nominal). The 72-hour ErC10 and EyC10 was determined to be 2.82 mg/L and 3.43 mg/L (nominal), respectively.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata was determined according to OECD TG 201, EU Method C.3 and the principles of GLP. The study was conducted under static conditions with an initial cell density of 6442 cells/mL. Five concentrations were tested in a geometrical series with factor of 2.5: 0.768, 1.92, 4.80, 12.0, 30.0 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The test item concentrations in the test media were confirmed by HPLC-DAD analysis to be within ± 20% of the nominal concentrations. Therefore, all effect values are based on the nominal test item concentrations. In result, the 72-hour ErC50 was 4.33 mg/L and the EyC50 was 3.93 mg/L. The 72-hour ErC10 and EyC10 was determined to be  2.82 mg/L and 3.43 mg/L (nominal), respectively. All validity criteria of the guidelines were fulfilled.

Description of key information

In a growth inhibition test with Raphidocelis subcapitata according to OECD TG 201, the 72-hour ErC50 was 4.33 mg/L and the EyC50 was 3.93 mg/L (nominal). The 72-hour ErC10 and EyC10 was determined to be  2.82 mg/L and 3.43 mg/L (nominal), respectively (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:

4.33 mg/L

EC10 or NOEC for freshwater algae:

2.82 mg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata was determined according to OECD TG 201, EU Method C.3 and the principles of GLP. The study was conducted under static conditions with an initial cell density of 6442 cells/mL. Five concentrations were tested in a geometrical series with factor of 2.5: 0.768, 1.92, 4.80, 12.0, 30.0 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The test item concentrations in the test media were confirmed by HPLC-DAD analysis to be within ± 20% of the nominal concentrations. Therefore, all effect values are based on the nominal test item concentrations. In result, the 72-hour ErC50 was 4.33 mg/L and the EyC50 was 3.93 mg/L. The 72-hour ErC10 and EyC10 was determined to be  2.82 mg/L and 3.43 mg/L (nominal), respectively. All validity criteria of the guidelines were fulfilled (reference 6.1.5-1).