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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:

Study was carried out between 6 and 9 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according OECD Guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of the signature 08.10.2010

Analytical monitoring:

yes

Details on sampling:

- Test water was sampled and pH and salinity were measured by the study laboratory.
- Sample storage conditions before analysis: Corresponding to the storage condition of test material.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions were prepared by the addition of a known quantity of mixed ash directly to 1 litre volumes of culture medium. These solutions were stirred for overnight using magnetic followers and then left to stand for 15 minutes. After this time the solutions were filtered through 0.45 µm cellulose nitrate filters, which had been previously washed with 100 mL Reverse Osmosis (RO) water.
- Controls: The control consisted of culture medium only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green alga Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): Brixham Environmental Laboratory culture from the strain
- Age of inoculum (at test initiation): A four day old culture of the alga in the exponential growth phase
- Method of cultivation: The culture was grown in the medium, and under the environmental conditions, described for the test.

ACCLIMATION
- Culturing media and conditions (same as test or not): The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium, the compounds of which are shown in the table 1.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Hardness:

NA

Test temperature:

22 ± 2°C.

pH:

7.6 - 7.7.

Dissolved oxygen:

NA

Salinity:

0 - 0.1‰.

Nominal and measured concentrations:

3.2, 5.6, 10, 18, 32 and 56 mg L-1

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks closed with foam bungs
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 ml, 100 mL of test solution
- Aeration: No
- Shaking: orbital shaking 160 rpm
- Initial cells density: 0.547 × 104 cells mL-1 (nominal cell density of 0.5 × 104 cells mL-1).
- No. of vessels per concentration (replicates): triplicate cultures of each concentration of mixed ash
- No. of vessels per control (replicates): Six replicate cultures of the culture medium control
- No. of vessels per vehicle control (replicates): One blank vessel (without algal inoculum) was incubated concurrently for each test concentration and the control.
- Blanks: One

GROWTH MEDIUM
- Standard medium used: AAP medium
- Detailed composition if non-standard medium was used: See Table 1.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH was measured at the start and at the end of the test from all vessels. Salinity was measuren corespondingly, but from three test concentrations ( 3.2, 18 and 56 mg L-1). The temperature of the incubator was continuously monitored using an electronic recording system with hourly recording of values. The light intensity was measured once during the study, in each of four representative positions, using a photometer reading in lux (cosine).

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted from 7.4 - 10.2 with 1 M hydrochloric acid to 7.4 - 7.5.
- Light intensity and quality: Continuous "cool-white" illumination of 6440 lux ± 13%

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter Coulter counter.
- Observation intervals: 24, 48 and 72 hours.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations: nominal mixed ash concentrations of 3.2, 5.6, 10, 18, 32 and 56 mg L-1

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Remarks on result:

other: p = 0.05

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Remarks on result:

other: p = 0.05

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Remarks on result:

other: 95% CL 8.10-8.77 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

6.52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Remarks on result:

other: 95% CL 5.74-6.92 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Remarks on result:

other: 95% CL 1.10-6.33 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: p = 0.05

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: p = 0.05

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 21.1-21.4 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 8.34-9.21 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 6.62-7.33 mg/L

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): At 32 mg L-1 some of the cells were observed to be enlarged, clumping and with ruptured cell walls. At 56 mg L-1 clumping of the cells was observed.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Biological data

Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass. The algal cell particle densities measured at each time period are given in Table 2. The means of these values are also shown in Table 2 and are plotted as growth curves in Figure 1.

Table 2 Algal cell densities

 

Nominal loading concentration of mixed ash (mg L^-1)	Replicate	Algal cell particle density (10^4 cells mL^-1)
		24 hours	48 hours	72 hours
Culture Medium	A	2.220	10.50	53.20
	B	2.320	12.60	57.90
	C	2.160	11.70	52.20
	D	2.410	12.60	54.80
	E	2.490	13.10	61.40
	F	2.290	12.00	57.40
	Mean	2.315	12.08	56.15
3.2	A	2.260	10.40	42.80
	B	2.290	12.50	58.90
	C	2.500	11.70	55.20
	Mean	2.350	11.53	52.30
5.6	A	2.380	11.80	50.20
	B	2.150	12.50	60.00
	C	1.810	11.40	48.80
	Mean	2.113	11.90	53.00
10	A	0.1980	2.080	14.10
	B	0.1840	2.480	18.00
	C	0.2700	2.610	16.20
	Mean	0.2173	2.390	16.10
18	A	0.2620	2.180	11.30
	B	0.1100	2.570	10.90
	C	0.08400	2.630	11.50
	Mean	0.1520	2.460	11.23
32	A	0.2240	0.00001	0.00001
	B	0.1800	0.00001	0.00001
	C	0.2240	0.00001	0.00001
	Mean	0.2093	0.00001	0.00001
56	A	0.1260	0.00001	0.00001
	B	0.00001	0.00001	0.00001
	C	0.00001	0.00001	0.00001
	Mean	0.04201	0.00001	0.00001

Yield This response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass.These cell particle densities were examined by one-way analysis of variance and Dunnett’s procedure was used to identify significant differences (p = 0.05) from the control. The test substance concentrations yields are given in Table 3, together with the yields expressed as percentages of thecontrol and significant differences from the control. A Weibull model was fitted to the percent effect and log concentration data. This model was then used to estimate the median effective concentration (based on yield curve area, E_yC50) and its 95% confidence limits.

Table 3 Mean yields over the test period

Nominal loading concentration of mixed ash (mg L^-1)	Mean yield (0 -72 hours)	Percentage of control
Control	55.60	-
3.2	51.75	93
5.6	52.45	94
10	15.55*	28
18	10.69*	19
32	-0.547*	-1
56	-0.547*	-1

* Significant difference (P=0.05) from the control

Growth rates

The growth rate (0 to72hours) was calculated for each replicate culture, according to the formula.

Growth rate = (Log_n(N₂/N₁))/t

where

N₁ = Cell density at start of test

N₂ = Celldensity at end of test

t = Time interval (days)

These data were analysed as described for the yield method. The mean growth rates,are given in Table 4, together with the rates expressed as percentages of the control.

A Weibull model was fitted to the percent effect and log concentration data. This model was then used to estimate the median effective concentration (based on yield curve area, E_yC50) and its 95% confidence limits.

Table 4 Mean growth rates over the test period

Nominal loading concentration of mixed ash (mg L^-1)	Mean daily growth (0 -72 hours)	Percentage of control
Control	1.544	-
3.2	1.520	98
5.6	1.525	99
10	1.127*	73
18	1.007*	65
32	-3.637*	-235
56	-3.637*	-235

* Significant difference (P=0.05) from the control

Physical and chemical data At the start of the test the pH ranged from 7.4 to 10.2, before adjustment, and 7.4 to 7.6, after adjustment, and at the end of the test the range was from 7.6 to 7.7 (Table 5). No pH shifts were observed in any sample. The hourly temperature measurements, recorded automatically, remained within 20±2°C. The mean light intensity calculated from a range of representative positions, measured once during the study, was 6440 lux (by cosine receptor), with a range of ±13%. The salinity of the test solutions, at the start and end of the test, was 0 to 0.1‰.

Table 5 Test solution pH and salinity measurements

Nominal loading concentration of mixed ash (mg L^-1)	Salinity %		pH
	0 hours	72 hours	0 hours		72 hours
			Before adjustment	After adjustment
Control	-	-	7.4	-	7.6
3.2	0.1	0.1	7.7	-	7.6
5.6	-	-	8.5	7.5	7.7
10	-	-	9.2	7.5	7.6
18	0.1	0.1	8.5	7.6	7.6
32	-	-	9.9	7.6	7.6
56	0.1	0.1	10.2	7.5	7.6

Validity criteria fulfilled:

yes

Conclusions:

The effect concentrations (EC50) for yield and growth were found to be 8.5 mg/L and 21.3 mg/L, respectively

Executive summary:

Short-term toxicity of Ash toward a green alga Pseudokirchneriella subcapitata was studied in a GLP compliant laboratory according to the OECD Guideline 201 (Alga, Growth Inhibition Test). The 72 h test was performed at six concentrations of filtered Ash solutions with three replicates in constant illumination and at temperature of 22 ± 2°C. The nominal cell density was 0.5 x 10⁴ cells. The growth curve for control was exponential. In the test, significant effects (p = 0.05) on alga growth was observed. The effect concentrations for yield and growth were found to be EyC50 = 8.5 mg/L and ErC50 = 21.3 mg/L, respectively.

Description of key information

Toxicity of Ash to Green alga Pseudokirchneriella subcapitata was determined in a GLP compliant laboratory study according to OECD Guideline 201 (Alga, Growth Inhibition Test ). The EC50 (72h) for growth rate was 21.3 mg/L and for yield it was 8.5 mg/L. 

Key value for chemical safety assessment

EC50 for freshwater algae:

21.3 mg/L

Additional information

Due to low water solubility of Ash, Water Accommondated Fractions of the test solutions were prepared.