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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:

26 Okt 2020 - 12 Apr 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Range finding:
- Concentrations: 0, 1.0, 10, 100 mg/L (nominal water accommodated fraction (WAF) loading rate)
- Sampling method: Water samples were taken from each WAF loading rate at 0 and 72 h for quantitative analysis on the day of sampling.
- Sample storage conditions before analysis: Analysis occurred on the day of sampling, unless the duplicate sample was analysed. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Definitive test:
- Concentrations: 0, 1.0, 3.2, 10, 32, 100 mg/L (nominal WAF loading rate)
- Sampling method: Water samples were taken from each freshly prepared bulk loading rate preparation at 0 h and from each loading rate after 72 h (replicates pooled). At 24 h and 48 h samples were taken from alongside samples.
- Sample storage conditions before analysis: Analysis occurred immediately, unless the duplicate sample was analysed. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Since the test substance is a complex mixture (UVCB) and is poorly soluble in water, organisms were exposed to water accommodated fractions (WAF) of the test item.
- Method: WAF

VALIDATION OF MIXING PERIOD:
- Loading rate: 100 mg/L (n=2)
- Stirring rate: stirring rate such that a vortex was formed to give a dimple at the water surface
- Duration of stirring: treatment 1: 23 h (n=1); treatment 2: 95 h (n=1)
- Standing periode: 1 h
- Sampling time: treatment 1: 24 h; treatment 2: 96 h
- Sampling method: The aqueous phase was removed by siphon.
- Test medium: AAP

Results showed that increasing the stirring period did not increase the amount of the test item in the WAF and so the stirring period of the WAF was maintained at 24 h.

PREPERATION OF WAF FOR THE RANGE FINDING TEST
- Loading rate WAF: 1.0, 10 and 100 mg/L (n=2)
- Description: Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2.0 and 2.0 L of culture medium.
- Stirring rate: stirring rate such that a vortex was formed to give a dimple at the water surface
- Duration of stirring: 23 h
- Standing periode: 1 h
- Test medium: AAP
- Sampling time: 24 h
- Sampling method: The aqueous phase was removed by mid-depth siphoning (the first 75 to 100 ml were discarded).
- Test concentration separation factor: 10
- Evidence of undissolved material: Microscopic observations of the WAFs showed no presence of micro-dispersions of the test item in all loading rates.

PREPERATION OF WAF FOR THE DEFINITIVE TEST
- Loading rate WAF: 1.0, 3.2, 10, 32 and 100 mg/L (n=3)
- Controls: 0 mg/L (n=6)
- Description: Nominal amounts of the test item were each separately added to the surface of 10, 5.0, 5.0, 5.0 and 5.0 L of test water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively.
- Stirring rate: stirring rate such that a vortex was formed to give a dimple at the water surface
- Duration of stirring: 23 h
- Standing period: 1 h
- Test medium: AAP
- Sampling time: 24 h
- Sampling method: The aqueous phase was removed by mid-depth siphoning (the first 75 to 100 ml were discarded).
- Test concentration separation factor: approx. 3.2
- Evidence of undissolved material: Microscopic observations of the WAFs showed no presence of micro-dispersions of the test item in all loading rates of the definitive test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source: In house culture, obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): 3 to 4 days
- Method of cultivation: Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at an initial cell density of approximately 10E3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E5 to 10E6 cells/mL. Pre-culture conditions gave an algal suspension in log phase growth.

ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

6.9 - 9.3

Nominal and measured concentrations:

Nominal: 0 mg/L and 1.0, 3.2, 10, 32 and 100 mg/L (WAF loading rate)
Measured (after 72 h): < LOD and 0.102, 0.235, 0.507, 0.862 and 2.03 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): The flasks were plugged with polyurethane foam bungs.
- Fill volume: 100 mL
- Aeration: no
- Rotation: 150 rpm
- Initial cells density: 5 x 10E3 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: AAP

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The cultrue medium was prepared using reverse osmosis purified deionized water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH of the control and each test concentration (0 h and 72 h); temperature within the incubator (daily)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: approx. 7000 lux, provided by warm white lightning (380-730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell densities were determined at 24, 48 and 72 h using a Coulter® Multisizer Particle Counter
- Microscopical inspection of test and control cultures after 72 h of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 3.2
- Range finding study : yes, 0, 1.0, 10 and 100 mg/L (WAF loading rate); The results showed no effect on growth at 1.0 mg/L loading rate WAF. However, growth was observed to be reduced at 10 and 100 mg/L loading rate WAF.
- Test concentrations: 0 mg/L and 1.0, 3.2, 10, 32 and 100 mg/L (WAF loading rate)

CULTURING APPARATUS
- Incubator: INFORS® Multitron incubator

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence limits 33 to 46 mg/L WAF loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

86 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence limits 82 to 90 mg/L WAF loading rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Details on results:

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 9.0 at 72 hours.

MICROSCOPIC INVESTIGATIONS - DEFINITIVE TEST
After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2, 10 and 32 mg/L loading rate WAF, however no intact cells were observed to be present in the test cultures at 100 mg/L loading rate WAF.

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- ErC50 (72 h): 1.3 mg/L
- Other: Performed twice in a 12 month period. Results of the most recent positive control are given. In-house data from the previous five studies shows a mean ErC50 value of 1.4 mg/L (standard deviation = 0.16) and a mean EyC50 value of 0.64 mg/L (standard deviation = 0.12)

Reported statistics and error estimates:

Prior to determination of the Lowest Observed Effect Concentration (LOEC) and No Observed Effect Concentration (NOEC), data for each endpoint was assessed using ShapiroWilk’s test on Normal Distribution, Levene’s test or Cochran’s test on Variance Homogeneity and trend Analysis by Contrasts (as necessary) to enable the correct comparison to be performed. The Williams Multiple Sequential t-test Procedure was used for yield and the Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm Procedure was used for growth rate. All statistical analyses were performed using the ToxRat computer software package (ToxRat® Solutions GmbH).

VALIDATION OF MIXING PERIOD

Table 1 Validation of mixing period

Nominal Loading Rate (mg/L)	Time (Hours)
	24	96
	Measured Concentration (mg/L)	Measured Concentration (mg/L)
100	21.9	20.0

It is evident from this work that increasing the stirring period did not increase the amount of dissolved test item in the WAF and so preparation of the WAF was maintained at 24 hours.

CHEMICAL ANALYSIS - RANGE FINDING TEST

 Chemical analysis of the test preparations at 0 hours showed measured concentrations to range from 1.2 to 64 mg/L and 72 hours showed measured concentration to range from 0.084 to 5.9mg/L indicating that the test item was not stable under test conditions.

INHIBITION OF GROWTH RATE - DEFINITIVE TEST

Table 2 Inhibition of growth rate in the definitive test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)
		0 to 72 Hour	% Inhibition
Control	R1	0.070	-
	R2	0.068
	R3	0.072
	R4	0.071
	R5	0.068
	R6	0.065
	Mean	0.069
	SD	0.003
1.0	R1	0.069	0
	R2	0.069	0
	R3	0.070	[1]
	Mean	0.069	[0]
	SD	0.001
3.2	R1	0.065	6
	R2	0.072	[4]
	R3	0.071	[3]
	Mean	0.069	[0]
	SD	0.004
10	R1	0.069	0
	R2	0.065	6
	R3	0.068	1
	Mean	0.067	2
	SD	0.002
32	R1	0.065	6
	R2	0.065	6
	R3	0.066	4
	Mean	0.065	5
	SD	0.001
100	R1	0.028	59
	R2	0.028	59
	R3	0.025	64
	Mean	0.027	61
	SD	0.002

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R = Replicate

SD = Standard deviation

[ ] = Increase in growth compared to controls

VALIDATION CRITERIA - DEFINITIVE TEST

Table 3 Validity criteria for OECD 201 (2006)

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.	The cell concentration of the control cultures increased by a factor of 144 in 72 h.	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%	12%	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%.	4%	yes

 

CHEMICAL ANALYSIS - DEFINITIVE TEST

Table 4 Analytical results – definitive test

Sample description	Time point (h)	Analyte detected (mg/L)	Nominal recovery (% LR)
Control	0	< LOD	-
1.0 mg/L LR WAF		0.0317	3
3.2 mg/L LR WAF		0.0801	3
10  mg/L LR WAF		0.111	1
32  mg/L LR WAF		15.7	49
100 mg/L LR WAF		18.3	18
1.0 mg/L LR WAF	24	0.0310	3
3.2 mg/L LR WAF		0.0606	2
10  mg/L LR WAF		0.156	2
32  mg/L LR WAF		1.73	5
100 mg/L LR WAF		3.86	4
1.0 mg/L LR WAF	48	0.177	12
3.2 mg/L LR WAF		0.245	8
10  mg/L LR WAF		0.678	7
32  mg/L LR WAF		1.94	6
100 mg/L LR WAF		3.02	3
Control	72	< LOD	-
1.0 mg/L LR WAF		0.102	10
3.2 mg/L LR WAF		0.235	7
10  mg/L LR WAF		0.507	5
32  mg/L LR WAF		0.862	3
100 mg/L LR WAF		2.03	2

 

Procedural recovery results for definitive test (1 and 100 mg/L, nominal, n=2 for each time point): 83 - 106%

 

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

Short-term toxicity to aquatic algae:

ErL50 (72 h) = 86 mg/L for Raphidocelis subcapitata (nominal, WAF loading rate, OECD 201)

Long-term toxicity to aquatic algae:

ErL10 (72 h) = 40 mg/L for Raphidocelis subcapitata (nominal, WAF loading rate, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

86 mg/L

EC10 or NOEC for freshwater algae:

40 mg/L

Additional information

The available key study on the toxicity of the UVCB test substance to the aquatic freshwater algae Raphidocelis subcapitata was performed according to OECD guideline 201 (GLP). Algae were exposed to individually prepared water accommodated fractions (WAF) of the UVCB test substance of 0, 1.0, 3.2, 10, 32 and 100 mg/L (nominal, WAF loading rate) for 72 h. Analytical determination of test solution concentration was performed by analyzing fatty acid concentrations in the WAFs as an surrogate for the UVCB test substance. Chemical analysis resulted in low concentrations of fatty acids of 0.0317 – 18.3 mg/L in the test medium at test initiation and 0.102 – 2.03 mg/L at test termination after 72 h. Since the toxicity cannot be linked to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only. Concentration dependent effects on the growth rate were observed after 72 h, resulting in a 72 h ErL50 of 86 mg/L (nominal, WAF loading rate) and a 72 h ErL10 of 40 mg/L (nominal, WAF loading rate).