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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:
2014-05-13 to 2014-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 201 with GLP compliance. All validity criteria were fulfilled and no deviations were observed.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on September 14, 2010 / signed on February 07, 2011
Specific details on test material used for the study:
No additional information
Analytical monitoring:
yes
Details on sampling:
To assess the initial concentrations of the test item, all test solutions were sampled prior to the beginning of the exposure periods and the addition of the algae.
After 24, 52 and 72 h, 20 mL samples for chemical analysis were taken from representative replicates of each treatment level.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The highest test concentration of 9.02 mg/L was prepared by pipetting 10 μL of test substance (specific density of 0.902 g/mL) in 1 L of sterile growth medium in a sterile 1 litre glass flask. The second highest test concentration was prepared by pipetting 5 μL of test substance in 1 L of sterile growth medium in a sterile 1 L glass flask. For pipetting micropipettes with a low dead-volume were used. The test solutions were stirred at about 100 rpm for 24 h. The 5 μL/L test solution was then diluted with sterile growth medium to obtain the other three test concentrations.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrechtvon Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG.
- Cultivation and pre-culture: The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980)). Prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test. The culture duration of the pre-cultures was 3 days.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
22.0-22.1 °C
pH:
The pH of the controls showed a slight increase during the test (8.04 at test start and 9.73 at test end). In the test cultures the initial pH ranged between 7.94 and 7.99 and between 7.91 and 9.53 at the end of the test.
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: 0.564, 1.13, 2.26, 4.51 and 9.02 mg/L
Measured concentrations: see "Any other information on results incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical glass flasks sealed with glass stoppers
- Material, size, headspace, fill volume: Every flask was filled with 100 mL of the respective test medium
- Type (delete if not applicable): Closed; test vessels were sealed with glass stoppers to prevent a possible volatilisation of the test item from the vessels.
- Initial cells density: The algal pre-culture (1510 μL at a cell density of 3.31 x 10^5 cells/mL) was added to the test vessels to achieve the initial cell concentration of 5000 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes; sterilised synthetic OECD medium according to OECD 201 was used as growth medium

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Light intensity and quality: Light intensity ranged from 99.07 to 100.4 μmol/m2/ s1
- The cultures were oscillated by continuously stirring on a laboratory shaker with 150 rpm (Incubation Shaker Multitron®, INFORS, Switzerland).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell concentrations were determined using an electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany). The correctness of the electronic counts was checked by microscope counts following internal standard operation procedures. During the test, the cell concentrations were determined daily in samples taken directly from the test vessels. The cells were counted after 24, 52 and 72 h. This was already taken into account in the calculations and did not affect the results.

TEST CONCENTRATIONS
- Range finding study: In a non-GLP range-finder test three nominal test concentrations of 0.1, 1.0 and 5.0 mg/L were tested. Based on visual observations, the test item was soluble at these concentrations. The test item was not analysed. Inhibition of growth rate was 0.4, -4.4 and 4.6 % at test concentrations of 0.1, 1.0 and 5.0 mg/L after 72 h. Respective inhibitions of yield were 2.9, -22.3 and 20.3 %.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.71 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 5.04-6.61 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.38 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.98-1.75 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.59 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
There were concentration-dependent effects on the growth of the green alga at test concentrations higher than 0.59 mg/L. Corresponding microscopy revealed normal appearances of the algae cells despite increasing cell debris with increasing growth inhibition.
Results with reference substance (positive control):
- Results with reference substance valid
- The latest nominal ErC50 value of 3.78 mg/L (3.64 - 3.92 95% confidence limits) of May 2014 is in good agreement with the results of an international ring test with ErC50 of 3.38 ± 1.30 mg/L.
Reported statistics and error estimates:
The test results were statistically analysed to determine an EC50, EC20 and EC10, and the respective 95 % confidence intervals, using Probit-analysis assuming log-normal distribution of the values. Individual replicate responses were used for the regression analysis. The NOEC and LOEC values for growth rate and yield were determined by the Williams Multiple Sequential t-test using the computer program ToxRat Professional.

Table 6.1.5/1: Percent inhibition of growth rate and yield compared to controls after 72 h

 

Treatment (mg/L)

% Inhibition of growth rate

% Inhibition of yield

Control

0.00

0.00

0.59

3.06

11.7

1.13

4.18

15.1

2.03

15.0

45.1

4.70

50.5

87.9

7.96      

56.7

90.9

EC50 [mg/L]

5.71 (95% CI: 5.04-6.61)

2.21 (95% CI: 1.95-2.52)

EC10 [mg/L]

1.38 (95% CI: 0.98-1.75)

0.83 (95% CI: 0.61-1.02)

NOEC [mg/L]

0.590

<0.590

  

Validity of the test :

The test fulfills all validity criteria of the OECD guideline 201 as:

The cell number in the control cultures increased by a factor of 50.7 within the 72 h test period (validity criterion: > 16).

Evaluation of the sectional growth rates of the controls: The mean of the replicate coefficients of variations (CV %) in the section-by-section growth rate of controls was 34.4 % during the test period (validity criterion ≤ 35 %).

The coefficient of variation of average specific growth rate at test end in replicate control cultures was 1.7 % (validity criterion ≤ 10 %).  

Test item analysis:

Generally, the test item concentrations were stable during the test period. At test start, the measured concentrations were 0.590, 1.13, 2.03, 4.70 and 7.96 mg/L, corresponding to 105, 100, 89.7, 104 and 88.3 % of nominal. After one and two days the recoveries of the test item ranged between 82.3 and 103 % of the measured initial concentrations. After three days, at test end, the measured concentrations were 0.436, 0.966, 2.03, 4.54 and 7.64 mg/L, corresponding to 73.8, 85.5, 100, 96.7 and 96.0 % of the measured initial concentrations (77.2, 85.5, 89.9, 101 and 84.7 % of nominal).

Since the recovery of the test item was slightly below 80 % only at the lowest concentration at test end and the mean measured concentrations (0.506, 1.05, 2.05, 4.51 and 7.44 mg/L) ranged between 85.8 and 101 % of the initial values, the test evaluation was based on the initial measured concentrations (0.590, 1.13, 2.03, 4.70 and 7.96 mg/L).

Validity criteria fulfilled:
yes
Conclusions:
There were concentration-dependent effects on the growth of Pseudokirchneriella subcapitata. The ErC50 and ErC10 values for inhibition of growth rate were 5.71 and 1.38 mg/L, respectively.
Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test material at the nominal concentrations of 0.564, 1.13, 2.26, 4.51 and 9.02 mg/Lfor 72 h under static conditions.

The concentrations of test material in the test media were determined at the beginning of the exposure period and then daily during the three day exposure period using LC-MS. Generally, the test material concentrations were stable during the test period. At test start, the measured concentrations were 0.590, 1.13, 2.03, 4.70 and 7.96 mg/L, corresponding to 105, 100, 89.7, 104 and 88.3 % of nominal. After one and two days the recoveries of the test item ranged between 82.3 and 103 % of the measured initial concentrations. After three days, at test end, the measured concentrations were 0.436, 0.966, 2.03, 4.54 and 7.64 mg/L, corresponding to 73.8, 85.5, 100, 96.7 and 96.0 % of the measured initial concentrations. Since the recovery of the test item was slightly below 80 % only once at test end and the mean measured concentrations (0.506, 1.05, 2.05, 4.51 and 7.44 mg/L) ranged between 85.8 and 101 % of the initial values, the test evaluation was based on the initial measured concentrations (0.590, 1.13, 2.03, 4.70 and 7.96 mg/L).

There were concentration-dependent effects on the growth of Pseudokirchneriella subcapitata. The ErC50 and ErC10 values for inhibition of growth rate were 5.71 and 1.38 mg/L, respectively.

 

Description of key information

OECD 201, GLP, key study, validity 1:

72h-ErC50 (Pseudokirchneriella subcapitata) = 5.71 mg/L (95% CL: 5.04 -6.61 mg/L) based on initial measured concentrations.

72h-ErC10 (Pseudokirchneriella subcapitata) = 1.38 mg/L (95% CL: 0.98 -1.75) based on initial measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
5.71 mg/L
EC10 or NOEC for freshwater algae:
1.38 mg/L

Additional information

One key study is available to assess the toxicity of the registered substance to aquatic algae, Pseudokirchneriella subcapitata. This study was performed according to the OECD Guideline 201 with GLP compliance, under static conditions for 72 hours.

The nominal registered substance concentrations were tested at 0.564, 1.13, 2.26, 4.51 and 9.02 mg/L. The concentrations of test material in the test media were determined at the beginning of the exposure period and then daily during the three day exposure period using LC-MS. Generally, the test material concentrations were stable during the test period. At test start, the measured concentrations were 0.590, 1.13, 2.03, 4.70 and 7.96 mg/L, corresponding to 105, 100, 89.7, 104 and 88.3 % of nominal. After one and two days the recoveries of the test item ranged between 82.3 and 103 % of the measured initial concentrations. After three days, at test end, the measured concentrations were 0.436, 0.966, 2.03, 4.54 and 7.64 mg/L, corresponding to 73.8, 85.5, 100, 96.7 and 96.0 % of the measured initial concentrations. Since the recovery of the test item was slightly below 80 % only once at test end and the mean measured concentrations (0.506, 1.05, 2.05, 4.51 and 7.44 mg/L) ranged between 85.8 and 101 % of the initial values, the test evaluation was based on the initial measured concentrations (0.590, 1.13, 2.03, 4.70 and 7.96 mg/L). There were concentration-dependent effects on the growth of Pseudokirchneriella subcapitata. The ErC50 and ErC10 values for inhibition of growth rate were 5.71 mg/L (95% CL: 5.04 -6.61 mg/L) and 1.38 mg/L (95% CL: 0.98 -1.75 mg/L), respectively.