Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

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:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with some non-standard elements. Results at the 70-h time point are valid, results at the 95-hour time point are considered not valid, because the pH deviated by more than 1.5 units.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
other: EU C.3
Qualifier:
according to
Guideline:
other: EPA Guideline EG8
Principles of method if other than guideline:
The test was extended to 4 days according to the EPA Guideline EG8 requirements.  Modifications of the test design were implemented to allow for a closed-system test.  A reduced initial algal density (1x10E3 cells/mL), an increased NaHCO3 concentration (300 mg/L), the addition of Fe-citrate and a reduced medium pH (7.0) were utilized in order to avoid substantial pH changes or carbon limited growth in the closed test vials.  pH was adjusted to 7 using HCl.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: The concentrations in the test medium were measured at the start and at the end of the test (t=95 hours). Duplicate samples were taken at the start of the test from test vials containing the test substance concentrations of 0, 0.33, 1.0, 1.8 and 3.3 mg/L (nominal), without algae. At the end of the test single samples were taken from two vials per test substance concentration of the same concentration series, containing algae.
Samples of 16 mL were taken by syringe from the test vials. Each sample was injected through the septum of a 40 mL sample vial containing 7 mL hexane, 16 mL of a 120 g/L salt (NaCl) solution and a magnetic stirrer. One sample series was transferred to the Residue Analysis Department (Zeist) to be analysed; the other series was stored in a refrigerator as a spare.

The sampling method is assumed to have avoided collection of any undissolved material that may have been present.
Vehicle:
yes
Details on test solutions:
- Controls: Algal growth medium

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tertiary butyl alcohol (TBA) was used as the carrier solvent. 

- An amount of 329.1 mg of the test substance was dissolved in 10 ml tertiary butanol (TBA). Dilutions were then prepared in TBA so as to yield a test substance concentration series of (0.10, 0.33, 1.0, 1.8 and 3.3) x 10E4 mg/L.

- Four µl of each test substance dilution in TBA was injected through the septum of the appropriate vials using a syringe into 40 ml of algal growth medium. The resulting test substance concentration series was 0, 0.11, 0.34, 1.1, 3.6 and 11.2 mg/L.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Source (laboratory, culture collection): The fresh-water green alga Selenastrum capricornutum (CCAP 278/4)1), which belongs to the order of Chlorococcales (class Chlorophyceae), was used as the test organism. The culture was supplied by the CCAP, The Freshwater Biological Association, the Ferry House, Far Sawrey, Ambleside, Cumbria LA22 OLP, England.

A preculture of algae in the exponential growth phase was prepared as detailed in OECD Guideline no. 201, and in accordance with EPA requirements it was sub-cultured two times during 4 days using the medium

- Method of cultivation: A preculture of algae in the exponential growth phase was prepared as detailed in OECD Guideline no. 201, and in accordance with EPA requirements it was sub-cultured two times during 4 days using the mediumprepared from concentrated stock solutions in ultra pure water. It was sterilized by micropore filtration and contained 300 mg/L NaHCO3 (not 50 mg/L as specified in the OECD Guideline). This in order to improve the buffer capacity of the medium especially when is culturing algae in a closed system. Furthermore, the medium contained Fe-citrate, because the growth of the algae would be erratic in the absence of complexed iron. The pH was adjusted to pH 7 using a HCl-solution.
Test type:
other: sealed system
Water media type:
freshwater
Limit test:
no
Total exposure duration:
95 h
Hardness:
No data
Test temperature:
24ºC+/-2ºC
pH:
After 70 hours of incubation the pH in the test media cultures ranged from pH 7.4 – 7.9 (pH 7.7-7.9 in the control and solvent control), whereas the pH of the test solutions without algae were found to be pH 6.3.
After 95 hour of incubation the pH in the test media cultures ranged from pH 8.4 - 10.0 (pH 9.5 - 10.0 in the control and solvent control), and in the corresponding solutions without algae, pH 6.9-7.0. In the opinion of the present reviewers, the elevated pH in test and control cultures at the 95-h time point makes it particularly difficult to interpret the dose-response and significance.
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations:  0 (Control), 0.10, 0.33, 1.0, 1.8 and 3.3 mg/L. It is possible that the limit of solubility may have been reached or exceeded in the upper test concentrations in this range.

Corresponding geometric mean measured concentrations: Not determined, Not determined, 0.045, 0.15, 0.28 and 0.55 mg/L

Chemical analysis: Measured concentration at the start of the test were found to be an average of 20.3+/- 1.2% of the nominal concentrations at the three highest concentrations. At the end of the test, the test concentrations were found to have decreased. The study report states that the concentrations are geometric mean measured concentrations based on analysis at the 0-h and 95-h time points. However, at the 70-h NOEC (0.1 mg/l, nominal) loading level, no analytical results are reported. The geometric mean concentrations are based on the overall average recovery for the three highest loading rates. The recovery at the 0.33 mg/l nominal concentration was excluded from the averaging.

Measured concentrations at the 70-h time point are not available, though at the shorter duration it is perhaps likely that levels of HMDS would have been higher than those measured at 95-h.

The results were interpreted with reference to mean measured concentrations.
Details on test conditions:
TEST SYSTEM

- Modifications to standard test: The test was extended to 4 days according to the EPA Guideline EG8 requirements.  Modifications of the test design were implemented to allow for a closed-system test. 

- Test vessel: Due to the high volatility of the test substance, the test was conducted in closed, silanized, 40 mL EPA vials with Teflon lined septum caps.  

- Test medium: The test medium was prepared from concentrated stock solutions in ultra pure water, sterilized by micropore filtration, containing 300 mg/l NaHCO3, in order to improve the buffering capacity of the medium since the test was run in a closed system.

- Initial cells density: A reduced initial algal density (1x10E3 cells/mL), an increased NaHCO3 concentration (300 mg/L), the addition of Fe-citrate and a reduced medium pH (7.0) were utilized in order to avoid substantial pH changes or carbon limited growth in the closed test vials.  

- The test was carried out in triplicate with three controls containing algae only, three controls containing algae and carrier solvent (TBA) and a single background concentration series containing test substance without algae


OTHER TEST CONDITIONS

- pH adjustment: pH was adjusted to 7 using HCl.

- Light source: fluorescent lamps

- Photoperiod: Continuous

- Light intensity: 78 to 86 µmol/s/m2


OBSERVATIONS

- Cell density measurements: The initial density was calculated from the measured density in the algal pre-culture used for the inoculation, divided by the appropriate dilution factor. After 23, 43.5, 70 and 95 hours of incubation, algal densities (cells/mL) and algal biovolume (µm3/mL) were determined with the electronic particle counter (Coulter Multisizer IIe). For each measurement a separate vial was sacrificed. Measured values were corrected for the background values in the appropriate blanks.

- Post-exposure observations: In order to test whether or not the effects seen were algistatic or algicidal, cultures containing 0 and 3.3 mg/L (nominal) test substance were diluted with fresh medium and incubated for 7 and 10 days.
Reference substance (positive control):
yes
Remarks:
 K2Cr2O7 and/or 3,5 dichlorophenol.
Duration:
95 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
95 h
Dose descriptor:
LOEC
Effect conc.:
0.28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
95 h
Dose descriptor:
EC10
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
95 h
Dose descriptor:
EC50
Effect conc.:
> 0.55 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
95 h
Dose descriptor:
EC10
Effect conc.:
0.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
95 h
Dose descriptor:
EC50
Effect conc.:
0.22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
95 h
Dose descriptor:
EC90
Effect conc.:
> 0.55 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
70 h
Dose descriptor:
EC50
Effect conc.:
> 0.55 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
70 h
Dose descriptor:
EC50
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
70 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
70 h
Dose descriptor:
EC10
Effect conc.:
0.09 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
70 h
Dose descriptor:
NOEC
Effect conc.:
0.01 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: This NOEC is reported in the study report, however there is significant uncertainty associated with this value.
Details on results:
- Exponential growth in the control (for algal test): yes

- At the end of the incubation, growth in the 3.3 mg/l flasks was higher than in the control, confirming the algistatic nature of the test substance.

- 70 h results should be used, as 95 h pH deviates by > 1.5 units.

- ErC50 result of >0.55 mg/l has been extrapolated to 1.3 mg/l (0.98 -1.82 mg/l). The limit of solubility for the test substance is 0.93 mg/L, therefore ErC50 is greater than the water solubility.
Reported statistics and error estimates:
EbC and ErC were calculated by means of a parametric model developped by Kooijman. EbC were calculated as recommended in the OECD guideline.

NOEC (Calculated no-effect-concentration) at 95 hours (model calculations using the DEBtox software)

Table 1. Results of analysis of test media without algae at start of test and with algae at end of test

Nominal concentration (mg/L)

Measured concentration at start of test (mg/L)

Percentage of nominal

Measured concentration at end of test (mg/L)

Percentage of nominal  

Geometric mean measured concentration (mg/L) 

0 (Control)

0

 

0

 

 

0.1

Not analysed

-

Not analysed

-

-

0.33

0.23

69.2

0.01

4.1

0.045

1.0

0.21

21.3

0.06

5.5

0.15

1.8

0.34

18.9

0.19

10.4

0.28

3.3

0.68

20.7

0.72

21.9

0.55

Average (excluding 0.33 mg/L nominal)

 

20.3

 

12.6

 

Table 2. Areas under the growth curves (A) and percentage reductions (IA) in each treatment

Parameter

Nominal concentration of hexamethyldisiloxane (mg/L)

Control

TBA Control

0.10

0.33

1.0

1.8

3.3

A t=0-70h

489

437

426

323

257

184

106

IA

0

0

8

30

44

60

77

A t=0-95h

2151

2076

2002

1904

1373

860

530

IA

0

0

5

10

35

59

75

Table 3.Average-specific growth rates and percentage inhibition in each treatment

Nominal concentration of hexamethyldisiloxane (mg/L)

Growth rate  (per day) (3d)

% inhibition (3d)

Growth rate  (per day) (4d)

% inhibition (4d)

Mean inhibition %

3d

4d

Control

1.98

-2.3

1.70

3.0

 

 

Control

1.98

-2.3

1.77

-1.1

 

 

Control

1.94

-0.1

1.77

-1.2

0.0

0.0

TBA Control

1.97

-1.6

1.73

1.5

 

 

TBA Control

1.91

1.4

1.79

-1.9

 

 

TBA Control

1.85

4.8

1.76

-0.2

 

 

0.10

1.95

-0.4

1.75

0.2

 

 

0.10

1.83

5.5

1.72

1.6

2.0

0.3

0.10

1.92

0.9

1.77

-0.8

 

 

0.33

1.69

12.7

1.74

0.8

 

 

0.33

1.78

8.4

1.76

-0.7

8.4

-0.8

0.33

1.86

4.1

1.79

-2.3

 

 

1.0

1.70

12.4

1.65

5.7

 

 

1.0

1.59

17.8

1.70

3.0

12.5

4.6

1.0

1.80

7.3

1.66

5.0

 

 

1.8

1.65

14.9

1.47

16.2

 

 

1.8

1.49

23.2

1.55

11.5

18.3

12.5

1.8

1.61

16.9

1.58

9.7

 

 

3.3

1.25

35.7

1.29

26.2

 

 

3.3

1.20

38.0

1.21

30.7

31.6

21.8

3.3

1.53

21.1

1.61

8.3

 

 

Table 4. Test results (based on the geometric mean measured concentrations)

Parameter

70 hours

95 hours

NOEC

0.01 mg/l

0.15 mg/l

NEC

n.d.

0.05 mg/l

EbC10

0.01 mg/l

0.05 mg/l

EbC50

0.18 mg/l

0.22 mg/l

EbC90

>0.55 mg/l

>0.55 mg/l

ErC10

0.09 mg/l

0.14 mg/l

ErC50

>0.55 mg/l

>0.55 mg/l

ErC90

>>0.55 mg/l

>>0.55 mg/l


Validity criteria fulfilled:
yes
Conclusions:
A 70-hour ErC50 value of >0.55 mg/L, ErC10 value of 0.09 mg/L and NOEC of 0.1 mg/L have been determined for the effects of the test substance on growth rate of Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata). However, given the uncertainty in the calculation of the NOEC, the present reviewers consider it is not a reliable result of the test, therefore the ErC10 value is considered to be the most useful long term test result from this study.

Description of key information

A 70-hour ErC50 value of >0.55 mg/l and ErC10 value of 0.09 mg/l (based on the geometric mean of measured concentrations) have been determined for the effects of the test substance on growth rate of Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata).

Key value for chemical safety assessment

EC50 for freshwater algae:
0.55 mg/L
EC10 or NOEC for freshwater algae:
0.09 mg/L

Additional information

A 70-hour ErC50 value of >0.55 mg/l and ErC10 value of 0.09 mg/l were determined in the key study which is considered reliable with restrictions. The restrictions concern the biological variability, pH change in test and control cultures, and issues with analytical recoveries. In view of the duration of the exposure period it is likely that the test organisms were exposed primarily to the parent form of the tested substance, although losses were observed by the end of the study. A NOEC of 0.01 mg/l was also reported but is not considered to be reliable, given the considerable variability in biological responses seen in the study. Results at the 95-hour time point are considered not valid, because the pH deviated by more than 1.5 units.