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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:

04 Feb - 12 Feb 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)

Version / remarks:

adopted March 2006, corrected July 28, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from all test item concentrations and from the control
- Sampling method: Duplicate samples were taken from the freshly prepared test media at test start. At test end the contents of test beakers of each treatment were poured together and duplicate samples were taken.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: Defined amounts of the test item were added directly to the test water for each test concentration and were carefully stirred for 24 h in the dark to dissolve as much of the test item as possible. After cessation of mixing and a following period (1 h) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration. Each concentration was prepared separately.
- Controls: Yes, medium without algae

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen, Göttingen, Germany.
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: pre-culture

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

21.8 to 22.7 °C

pH:

8.1 to 10.1 (Control)
7.8 to 10.0 (Test item concentrations)

Nominal and measured concentrations:

100, 31.6, 10.0, 3.2 and 1.0 mg/L (nominal test item concentrations)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (loosely covered)
- Material, size, headspace, fill volume: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium
- Initial cells density: 0.5 x 10E04 algal cells per mL test medium
- Control end cells density: 121.6 x 10E04 algal cells per mL test medium
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Analytical grade salts were added at the nominal concentrations in deionised water
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: 6048 lux (range: 5820 to 6320 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometric measurement.The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range-finding test: Range-finding test was performed under non-GLP conditions

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

43.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

22.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No remarkable observations, clear test medium

Results with reference substance (positive control):

For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions.
EC50 (72 h) for growth rate: 1.11 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by the three parametric normal concentration distribution function (CDF).
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Dunnett's t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Table 1: Cell numbers

Nominal loading rate	Flask No.	Density of algal cells [10000/mL] after
[mg test item/L]		24 h	48 h	72h
Control	1	3.917	25.443	110.325
	2	3.736	24.320	120.327
	3	4.099	27.877	130.746
	4	3.917	25.069	123.036
	5	3.917	23.945	118.452
	6	3.736	25.630	126.787
	M	3.887	25.381	121.612
	S	0.137	1.384	7.089
1.0	1	3.917	24.507	117.410
	2	3.554	23.009	110.325
	3	4.099	23.945	110.950
	M	3.857	23.821	112.895
	S	0.278	0.757	3.922
3.2	1	4.645	23.197	109.700
	2	4.463	20.763	96.781
	3	4.281	22.073	107.825
	M	4.463	22.011	104.769
	S	0.182	1.218	6.981
10.0	1	3.008	18.516	90.113
	2	3.554	18.142	90.946
	3	3.554	19.078	93.447
	M	3.372	18.579	91.502
	S	0.315	0.471	1.735
31.6	1	2.644	10.841	54.481
	2	2.826	9.905	53.022
	3	2.664	11.777	57.190
	M	2.705	10.841	54.898
	S	0.105	0.936	2.115
100	1	3.190	2.791	13.431
	2	1.917	2.042	14.473
	3	2.281	2.604	12.806
	M	2.463	2.479	13.570
	S	0.656	0.390	0.842

m: mean value; s: standard deviation; At test start nominal 5000 algal cells/mL were incubated

Table 2: Growth Rates µ and Percentage Inhibition of µ during the Test Period

Nominal loading rate	Growth rate µ [1/day] and % inhibition of µ
[mg test item/L]	0 - 24 hours		0 - 48 hours		0 – 72 hours
	µ	%	µ	%	µ	%
Control	2.050	-	1.963	-	1.831	-
1.0	2.041	0.4	1.932	1.6	1.806	1.3
3.2	2.188	-6.7	1.892	3.6 (*)	1.781	2.7 (*)
10.0	1.906	7.1	1.807	7.9 (*)	1.736	5.2 (*)
31.6	1.688	17.7 *	1.537	21.7 *	1.566	14.5 *
100	1.572	23.3 *	0.796	59.4 *	1.100	39.9 *

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control;

* mean value significantly different from the control(tested with Dunnett's t-test (24, 48 and 72h),a = 0.05, one-sided)
(*) statistically significant nut not considered to be scientifically relevant

 

Table 3: Summary of Analytical Results

Sample Description	%of nominal 1)
Loading rate	fresh (0 h)	aged (72 h)
	[%]	[%]
Control (untreated)	n.a.	n.a.
Dilution Solvent (analytical performance)	n.a.	n.a.
Control	n.a.	n.a.
1 mg/L Loading Rate	97	50
3.2 mg/L Loading Rate	89	58
10.0 mg/L Loading Rate	90	70
31.6 mg/L Loading Rate	61	56
100 mg/L Loading Rate	37	36

1) mean value of all measured samples per treatment group; RSD: relative standard deviation per treatment group; n.a.: not applicable

Table 4: 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.	243.2	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%	13.5 %	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 and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1.1 %	Yes

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

ErL50 (72 h) > 100 mg/L (Growth rate, OECD 201, Pseudokirchneriella subcapitata, nominal)

ErL10 (72 h): 22.8 mg/L (Growth rate, OECD 201, Pseudokirchneriella subcapitata, nominal)

Additional information

There is one experimental study available, in which the toxicity of the substance was assessed according to the OECD guideline 201 and GLP.In a static test, Pseudokirchneriella subcapitata was exposed to the five nominal loading rates of 1.0, 3.2, 10.0, 31.6, and 100 mg/L test item for 72 h (initial cell density: 0.5 * 10E+04 cells/mL). Since the substance is a UVCB, the test solution was applied as water accommodated fraction (WAF). The actual test item concentrations were analytically verified via quantification of one component of the UVCB for fresh and aged samples by HPLC- UV detection. After 72 h, a clear dose-response curve was obtained for inhibition of growth rate and yield. Based on the quantified UVCB component the test concentrations were found to decrease over the course of the 72 h test in all test concentrations, except for 100 mg test item/L. The initial measured concentrations ranged between 37 and 97% of nominal and the aged samples between 36 and 70% of nominal. Since the pH increased up to 10 during the experimental phase degradation via hydrolysis is potentially expected but due to a higher degradation for the fish study with a lower pH this reason for degradation via hydrolysis is not presumably. Biological significant effects were determined and resulted in EL10 (72 h) of 22.8 mg/L (nominal) and EL50 (72 h) of > 100 mg/L (nominal) based on growth rate. For yield statistically significant effects resulted in an EL10 (72 h) of 4.63 mg/L and EL50 (72h) of 25.4 mg/L. Since the test was dosed via the water accommodated fraction (WAF) approach, all reported results refer to loading rate.