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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 8 January 2012 and 5 February 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD Guideline and GLP
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Sponsor's identification: Lemonile
Description: clear colourless liquid
Purity: 99.9%
Batch number: PE00027352
Date received:03 October 2011
Expiry date: 26 October 2013
Storage conditions: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 96 hours for quantitative analysis. Duplicate samples were taken at each occasion and stored at approximately -20ºC for further analysis if necessary.
An additional test sample of each test concentration was prepared at 0 hours and incubated alongside the study to provide samples for analysis at 72 hours.
Given that chemical analysis of the range-finding test preparations at 96 hours showed a concentration dependant decline in measured test concentration occurred it was considered likely that the test item was adsorbing to the algal cells present. It was therefore considered appropriate to prepare additional test samples at the start of the test with the omission of algal cells. These samples were incubated alongside the test and taken for analysis at 72 and 96 hours.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
An amount of test item (1100 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 41 mg/l. A series of dilutions was made from this saturated solution to give further stock solutions of 13, 4.1, 1.3 and 0.41 mg/l. An aliquot (1500 ml) of each of the stock solutions was separately inoculated with 29.5 ml of algal suspension to give the required nominal test concentrations of 0.41, 1.3, 4.1, 13 and 41 mg/l (equivalent to 0-Hour measured test concentrations of 0.26, 1.1, 3.1, 14 and 44 mg/l).

The control group was maintained under identical conditions but not exposed to the test item.
Due to the possible light sensitive nature of the test item all test item preparation was performed under laboratory safety lighting/shielded from the light.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source: obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1°C. Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1C until the algal cell density was approximately 10^4 - 10^5 cells/ml

ACCLIMATION
- Culturing media and conditions: The culture medium used for the definitive test was the same as that used to maintain the stock culture with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).
Test type:
static
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24 ± 1°C
Nominal and measured concentrations:
0.41, 1.3, 4.1, 13 and 41 mg/l (nominal test concentrations)
0.26, 1.1, 3.1, 14 and 44 mg/l (0-Hour measured test concentrations)
Details on test conditions:
TEST SYSTEM
250 ml glass conical flasks were used. Six flasks each completely filled with test preparation were used for the control and three flasks each completely filled were used for each treatment group
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 5.09 x 105 cells per ml. Inoculation of 1500 ml of test medium with 29.5 ml of this algal suspension gave an initial nominal cell density of 1 x 104 cells per ml and had no significant dilution effect on the final test concentration


TEST MEDIUM / WATER PARAMETERS
The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
For the purposes of the definitive test, additional sodium bicarbonate (500 mg/l) was added to the prepared culture medium prior to use.


OTHER TEST CONDITIONS
The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 96 hours

EFFECT PARAMETERS MEASURED:
Samples were taken at 0, 25, 50, 72 and 96 hours and the cell densities determined using a Coulter® Multisizer Particle Counter

TEST CONCENTRATIONS
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.041, 0.41, 4.1 and 41 mg/l for a period of 96 hours.

Based on the results of the range-finding test the following nominal test concentrations were assigned to the definitive test: 0.41, 1.3, 4.1, 13 and 41 mg/l.
Reference substance (positive control):
yes
Remarks:
Zinc chloride
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 3.0 - 4.4 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 2.9 - 4.3 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.26 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Range-finding Test:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.
The results showed no effect on growth at the nominal test concentrations of 0.041 and 0.41 mg/l. However, growth was observed to be reduced at 4.1 and 41 mg/l.
Based on this information nominal test concentrations of 0.41, 1.3, 4.1, 13 and 41 mg/l were selected for the definitive test.


Definitive test:
Cell density values determined at each sampling time and pH values at 0 and 96 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4. Growth rate, yield and biomass integral values for the control and test cultures after 96 hours and percentage inhibition values are given in Table 5.

The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7


Re-growth test
Re-growth was observed to have occurred in the control, 0.26 and 1.1 mg/l test cultures after 24 hours, in the 3.1 mg/l test culture after 48 hours and in the 14 and 44 mg/l test cultures after 216 hours. These results indicate that the test item was algistatic in effect.
Results with reference substance (positive control):
A positive control (Harlan Laboratories Ltd Project No: 41202063) used zinc chloride as the reference item at concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/l. The study was conducted in completely filled and sealed 250 ml ground glass stoppered conical flasks, the culture medium used was prepared with the addition of 500 mg/l NaHCO3. Data evaluation for the positive control was performed as per the definitive study. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

Time Point Response EC50 95% CL NOEC LOEC
(Hours) Variable (mg/l) (mg/l) (mg/l) (mg/l)

72 Growth Rate 0.23 0.20-0.28 0.010 0.032
Yield 0.090 0.074-0.11 0.010 0.032
Biomass 0.12 0.11-0.15 0.032 0.10
96 Growth Rate 0.30 0.24-0.39 0.010 0.032
Yield 0.073 0.061-0.089 0.010 0.032
Biomass 0.096 0.074-0.12 0.010 0.032

Historical in-house data for the exposure of Pseudokirchneriella subcapitata to zinc chloride using standard exposure conditions (100 ml of test preparation in a 250 ml conical flask plugged with a polyurethane foam bung) gave the following results:


Time Point Response Mean EC50 Standard NOEC LOEC
(Hours) Variable (mg/l) Deviation (mg/l) (mg/l)

72 Growth Rate 0.48 0.16 0.10 0.32
Yield 0.26 0.069 0.10 0.32
Biomass 0.28 0.070 0.10 0.32
96 Growth Rate 0.50 0.13 0.10 0.32
Yield 0.26 0.054 0.10 0.32
Biomass 0.26 0.057 0.10 0.32

It is evident from these results that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed test vessels) has a slight effect on the EC50 values obtained and, has a notable effect on the No Observed Effect Concentration (NOEC). As such it can be considered that the results obtained from the definitive test give a worst case result for the test item.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).


Additional test samples were prepared at the start of the test with the omission of algal cells. These additional samples were incubated alongside the test and sent for analysis at 72 and 96 hours. Analysis of these samples showed that measured concentrations in the range of 80-120% of the 0-Hour measured test concentrations were obtained with the exception of the 1.3 mg/l test sample at 72 hours where a measured concentration of 77% of the 0-Hour measured concentration was obtained. 

Given this it was considered that the decline in measured test concentrations was due to adsorption of the test item to the algal cells present. As such it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations only which were determined to be 0.26, 1.1, 3.1, 14 and 44 mg/l.

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 96-Hour period and gave the following results based on the 0-Hour measured test concentrations:

TiPoint

Response Variable

EC50(mg/l)

95% Confidence Limits (mg/l)

No Observed Effect Concentration (NOEC) (mg/l)

Lowest Observed Effect Concentration (LOEC) (mg/l)

72

Growth Rate

3.6

3.0

-

4.4

0.26

1.1

Yield

1.8

1.5

-

2.0

0.26

1.1

Biomass

1.9

1.7

-

2.2

0.26

1.1

96

Growth Rate

3.6

2.9

-

4.3

1.1

3.1

Yield

1.7

1.5

-

1.9

0.26

1.1

Biomass

1.8

1.6

-

2.1

0.26

1.1

Validity criteria fulfilled:
yes
Conclusions:
The 72 h EC50 for growth rate was 3.6 mg/L (95% CL= 3.0 -4.4 mg/L). The 96 h EC50 for growth rate was 3.6 mg/L (95% CL = 2.9 -4.3 mg/L).
The 72 h NOEC for growth rate was 0.26 mg/L and the 96 h NOEC for growth rate was 1.1 mg/L

Description of key information

The 72 h EC50 for growth rate was 3.6 mg/L (95% CL= 3.0 -4.4 mg/L). The 96 h EC50 for growth rate was 3.6 mg/L  (95% CL = 2.9 -4.3 mg/L). The 72 h NOEC for growth rate  was 0.26 mg/L and the 96 h NOEC for growth rate was 1.1 mg/L .

Key value for chemical safety assessment

EC50 for freshwater algae:
3.6 mg/L

Additional information

The key study was performed according to GLP and OECD Guideline 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at nominal concentrations of 0.41, 1.3, 4.1, 13 and 41 mg/l for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Analysis of the test preparations at 72 hours showed a concentration dependant decline in measured test concentration in the range of less than the limit of quantitation (LOQ) of the analytical method employed at 0.41 mg/l through to 45 mg/l at the nominal concentration of 41 mg/l (9% to 101% of the 0-Hour measured test concentrations). A further decline in measured test concentration was observed at 96 hours in the range of less than the LOQ of the analytical method employed at 0.41 mg/l through to 43 mg/l at the nominal concentration of 41 mg/l (9% to 98% of the 0-Hour measured test concentrations). 

Chemical analysis of the range-finding test preparations had shown a concentration dependant decline in measured test concentration at 96 hours and hence it was considered likely that the test item was adsorbing to the algal cells present. As such additional test samples were prepared at the start of the test with the omission of algal cells. These additional samples were incubated alongside the test and sent for analysis at 72 and 96 hours. Analysis of these samples showed that measured concentrations in the range of 80-120% of the 0-Hour measured test concentrations were obtained with the exception of the 1.3 mg/l test sample at 72 hours where a measured concentration of 77% of the 0-Hour measured concentration was obtained. 

Given this it was considered that the decline in measured test concentrations was due to adsorption of the test item to the algal cells present. As such it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations only which were determined to be 0.26, 1.1, 3.1, 14 and 44 mg/l.

A re-growth test was performed which showed the test item to be algistatic in effect.

The preferred observational endpoint in this study is algal growth rate inhibition because it is not dependent on the test design (ECHA guidance Chapter R.7b v 1.1).