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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
72 hours
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to OECD guideline 201 (2006)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch number - LPBS2A2005Appearance- white, crystalline, odourless powder
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):Not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
At the start and at the end of the incubation period, samples of the test media were drawn and the concentrations of the test substance were determined in aliquots of the blank containing test substance, but no algae; of one replicate of each of the test and control cultures (the 72 hours samples were filtered through a 0.20 µm filter)One sample was taken of each of the above listed aliquots and immediately analysed in duplicate by HPLC

Test solutions

Vehicle:
no
Details on test solutions:
First experiment - A stock solution with a nominal test substance concentration of 111 mg/L was prepared by dissolving 111.4 mg test substance in 1 L of nutrient medium by stirring for 5 minutes. Aliquots of this stock solution were diluted with nutrient medium to obtain lower concentrations.Second experiment - A stock solution with a nominal test substance concentration of 111 mg/L was prepared by dissolving 111.2 mg test substance in 1 L of nutrient medium by manual homegenisation. The pH of the stock solution (4.05) was adjusted with 0.1 M NaOH to the pH of nutrient medium (7.46). Afterwards the stock solution was filtrated (0.2 µm, Minisart, Sartorius) to maintain sterile conditions.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) ATCC (American Type Culture Collection) 22662, obtained from LGC Promochem GmbH, Mercatorstraße 51, 46485 Wesel A Rhein, Germany.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Not applicable
Test temperature:
The temperature was stable throughout the test periods (22-23 °C) in both of the experiments.
pH:
First experiment: The pH was between 4.2 and 7.5 at the start of the incubation in the test cultures and it was 7.4 in the control cultures. After 72 hours of incubation the pH was between 4.3 and 7.6 in the test cultures and it was 7.4 in the control cultures.Second experiment: The pH was between 7.3 and 7.5 at the start of the incubation, the pH was 7.3 in the control cultures. At the end of the test the pH was between 8.4 and 9.5 in the test cultures and it was 9.2 in the control cultures.
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
First experiment - nominal concs. - control, 0.39, 0.78, 1.56, 3.13, 6.30, 12.5, 25, 50 and 100 mg/L. The limit of quantification of the analytical method was 3.55 mg/L, therefore the four lowest nominal concentrations could not be analysed. Geometric mean measured concs in the remaining test media were 6.20, 13.96, 23.5, 50.41 and 96.37 mg/LSecond experiment - nominal concs. - control, 0.39, 0.78, 1.56, 3.13, 6.30, 12.5, 25, 50 and 100 mg/L. The limit of quantification of the analytical method was 3.55 mg/L, therefore the four lowest nominal concentrations could not be analysed. Geometric mean measured concs in the remaining test media were 4.47, 11.9, 24.02, 45.96 and 110.47 mg/L
Details on test conditions:
At the onset of the experiments the cell concentration in the preculture was determined and the inoculum was prepared by diluting an appropriate volume of the preculture with nutrient medium to give a calculated cell concentration of 10exp5 cells/mL.All test cultures and the blanks were set up in 250 mL conical glass flasks (Erlenmeyer). The total culture volume was 100 mL.Each test substance culture consisted of 10 mL inoculum (10exp5 algae/mL) and of 90 mL of the respective test substance preparation.The blanks without algae contained 90 mL of the undiluted stock solution and 10 mL of nutrient medium.The negative control cultures contained 10 mL of inoculum and 90 mL of nutrient medium.All cultures and the blank were incubated at 21 - 24 (± 2) °C for three days in permanent light. In the first experiment the light intensity was between 5250 and 5980 lux with a maximum deviation from the mean light intensity of 8 %. In the second experiment the light intensity was between 6040 and 6960 lux with a maximum deviation of 7 %. For each experiment three replicate cultures were incubated for each test substance concentration and six replicates for the control.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
No change in the colour of the media and no precipitation of test substance was noted during the incubation period of the experiments.Microscopic evaluation of the inoculum at the start of the test revealed normal appearance of the algae in both experiments. At the end of the test morphological changes of the algae (disruption of cell membranes) were observed in the group containing the highest test substance concentration in the first experiment. All other test cultures showed normal appearance of the algae at the end of the test.
Results with reference substance (positive control):
The last reference test with K2Cr2O7 was conducted from the 21st to the 24th of June 2010 with Pseudokirchneriella subcapitata to evaluate the reliability of the test conditions. The 72 hour EC50 for growth rate and yield were 1.32 and 0.62 mg K2Cr2O7/L, respectively. These results establish the reliability of the test procedures for this kind of study type.
Reported statistics and error estimates:
The mean yield of the test cultures is expressed as percentage of the mean yield of the control cultures and from this the percentage inhibition in yield is calculated. The logarithms of the substance concentrations are then plotted against the corresponding percent inhibition. The EyC50 for the 72 h incubation period is taken from the intercept of this curve with the parallel drawn to the abscissa at 50 % inhibition.Based on the yield as well as on the average growth rates two "lowest observed effective concentrations" (LOECs) are calculated for each experiment by comparison of the data of the three replicates of each test substance culture with the negative control (analysis of variance, followed by the Scheffé-test, P = 0.05). The "no observed effect concentrations" (NOECs) are then derived from these results (highest concentration with no statistically significant difference to the control).

Any other information on results incl. tables

Measured concentrations in the two experiments

Nominal concentration (mg/L)

Measured concentrations (mg/L)

Geometric mean (mg/L)

0 hours

72 hours

                                                                                  First experiment

Control

<QL

<QL

-

0.39

 <QL

<QL

-

0.78

 <QL

<QL

-

1.56

<QL

 <QL

-

3.13

 <QL

<QL

-

6.30

6.77

5.36

6.02

12.5

13.88

14.05

13.96

25

25.18

21.93

23.50

50

50.75

50.07

50.41

100

99.08

93.73

96.37

100 (blank, no algae)

99.30

98.22

98.76

Second experiment

Control

<QL

<QL

-

0.39

 <QL

 <QL

-

0.78

 <QL

 <QL

-

 1.56

<QL

<QL

 -
 3.13

<QL

<QL

 -
 6.30  4.02 4.98  4.47 
 12.50 12.56  11.28   11.90
 25.0 23.64  24.41   24.02
 50.0  47.94 44.06   45.96
 100 123.11  99.12   110.47
 100 (blank, no algae) 97.87  88.98  93.32 

QL: Quantification limit of the analytical method used (355 mg/L)

Mean yield, average specific growth rates and percent inhibition

Nominal concentration (mg/L)

Yield (10000 cells/mL)
 0 72 hours

Growth rate (per day)
0 72 hours

% inhibition

Mean

SD

Mean

SD

Yield

Growth rate

First experiment

Control

763.2

167.9

1.44

0.07

-

-

0.39

1066.3

425.5

1.54

0.13

-39.9

-6.9

0.78

1032.4

220.5

1.54

0.07

-35.4

-7.0

1.56

692.1

275.6

1.40

0.15

9.2

3.1

3.13

727.2

232.5

1.42

0.12

4.6

1.5

6.3

883.8

85.3

1.50

0.03

-15.9

-3.8

12.5

950.0

439.7

1.50

0.15

-24.6

-4.0

25

1048.6

252.4

1.55

0.08

-37.6

-7.3

50

89.9

79.1

0.65

0.38

88.2

54.7

100

-0.8

6.8

-0.15

0.42

100.1

110.5

Second experiment

Control

1087.5

86.0

1.57

0.03

-

-

0.39

1224.9

145.9

1.60

0.04

-12.6

-2.5

 0.78  1328.4 175.4  1.63  0.04  -22.2  -4.2 
 1.56  1065.3 63.2  1.56  0.02  2.0  0.4 
 3.13  1188.2 80.6  1.59  0.02  -9.3 -1.9 
 6.3  1015.7  128.5  1.54 0.04  6.6 1.5 
 12.5  1109.0 218.4   1.57 0.07  -2.0  -0.2 
 25  938.6 97.4 1.52 0.04 13.7  3.1 
 50 1231.5   268.9  1.60 0.08  -13.2  -2.3 
 100 1234.7   184.6  1.61  0.05 -13.5  -2.6 

Negative figures for inhibition indicate algal growth.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Algae were exposed over a 72 hour period to succinic acid at nominal concentrations of 0 (control), 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L.The pH of the medium was ranged between 7.4 and 7.5 at the start of the exposure period.   Concentrations of succinic acid were verified analytically at test initiation and termination.  The limit of quantification for succinic acid was 3.55 mg/L, therefore the concentrations of the four lowest test media were not quantified.Geometric mean measured concentrations of the remaining test media were 4.47, 11.9, 24.02, 45.96 and 110.47 mg/L.Based on the nominal exposure concentration both the 72-hour EyC50and ErC50were determined to be >100 mg/L with corresponding NOECs of 100 mg/L. 
Executive summary:

Two 72 hour algal inhibition experiments were conducted with succinic acid and Pseudokirchneriella subcapitata according to the OECD 203 guideline.

In the first experiment algae were exposed over a 72 hour period to succinic acid at nominal concentrations of 0 (control), 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L. pH ranged from 4.2 in the 100 mg/L treatment to 7.5 in the 0.39 mg/L treatment in a dose response relationship.  Concentrations of succinic acid were verified analytically at test initiation and termination.  The limit of quantification for succinic acid was 3.55 mg/L, therefore the concentrations of the four lowest test media were not quantified. Geometric mean measured concentrations of the remaining test media were 6.02, 13.96, 23.5, 50.41 and 96.37 mg/L. Based on nominal exposure concentrations the 72-hour EyC50was determined to be 40.7 mg/L with a corresponding NOEC of with 25 mg/L. The 72-hour ErC50was determined to be 46.8 mg/L with a corresponding NOEC of with 25 mg/L. 

In the second experiment algae were exposed over a 72 hour period to succinic acid at nominal concentrations of 0 (control), 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L.

The pH of the medium was ranged between 7.4 and 7.5 at the start of the exposure period.   

Concentrations of succinic acid were verified analytically at test initiation and termination.  The limit of quantification for succinic acid was 3.55 mg/L, therefore the concentrations of the four lowest test media were not quantified.Geometric mean measured concentrations of the remaining test media were 4.47, 11.9, 24.02, 45.96 and 110.47 mg/L.

Based on the nominal exposure concentration both the 72-hour EyC50and ErC50were determined to be >100 mg/L with corresponding NOECs of 100 mg/L. 

Based on the fact that the effects seen in the first experiment were influenced by the pH change the most appropriate endpoint to use for the assessment of succinic acid toxicity is the 72 hour ErC50value of >100 mg/L obtained in the study with pH adjustment.