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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 january 2010 to 11 november 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to standardised guidelines.

Data source

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

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method:
For quantification of the actual test item concentrations, at least duplicate samples were taken from the test media of all treatments at the start of the test (samples without algae) and at the end of the study (with algae). For sampling at the end of the test, the test medium of all treatment replicates were pooled. Alternatively, additional flasks containing the test media with algae was incubated separately under the test conditions for analytical purposes.
- Sample storage conditions before analysis:
All samples were deep-frozen immediately after sampling and were stored at about -20 °C until analysis. Remaining samples were stored until submission of the final report to enable additional analyses, if requested by the Sponsor. After finalization of the report, all samples were discarded.
If the test item has been shown to be unstable under the storage conditions, the samples will be analyzed immediately after sampling without prior storage or measures will be taken (e.g. pH adjustment or addition of organic solvents) in order to maintain the stability of the test item until analysis.

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): A culture of Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) is obtained from a commercial culture collection. The algae are cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.
- Age of inoculum (at test initiation): An inoculum culture of algae was set up three days before the start of the test.
- Method of cultivation: under standardized conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg/L as CaCO3
Test temperature:
22°C
Nominal and measured concentrations:
The following nominal concentrations of the test item were tested: 1.0, 3.2, 10, 32 and 100 mg/L.
Measured concentrations: 0.54, 2.7, 9.7, 32 and 96 mg/L (Mean geometric)
Details on test conditions:
TEST SYSTEM
- Test vessel: 50-mL Erlenmeyer flasks were used.
- Type (delete if not applicable): closed; Each test flask was covered with a glass stopper.
- Material, size, headspace, fill volume: in 50-mL Erlenmeyer flasks, completely filled with 60 ml of test medium.
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: 10000 cells/mL
- Control end cells density: The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter, Model ZM).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
The test was performed in a closed system, the test water was modified according to the International Standard ISO 14442 [6]. The concentration of NaHCO3 was increased by 200 mg/L to 250 mg/L (as a carbon source for algal growth), and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) was added to keep the pH of the test media as constant as possible. Analytical grade salts were dissolved in sterile purified water to obtain the following concentrations:

Macro-nutrients
NaHCO3 250.0 mg/L
KH2PO4 1.6 mg/L
MgSO4 × 7 H2O 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
Trace elements
H3BO3 185.0 µg/L
MnCl2 × 4 H2O 415.0 µg/L
ZnCl2 3.0 µg/L
CoCl2 × 6 H2O 1.5 µg/L
CuCl2 × 2 H2O 0.01 µg/L
Na2MoO4 × 2 H2O 7.0 µg/L
FeCl3 × 6 H2O 64.0 µg/L
Na2EDTA × 2 H2O 100.0 µg/L

The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).

- Culture medium different from test medium: no
- Intervals of water quality measurement: The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: The pH was measured and recorded in each treatment at the start and at the end of the test.
- Light intensity and quality: The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7900 Lux (range: 6990 to 8440 Lux, measured at nine places in the experimental area). The light intensity over the incubation area was within ±15% from the average light intensity as recommended by the guideline.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

- Range finding study: yes
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
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:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 96 mg/L
Nominal / measured:
meas. (geom. mean)
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:
The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) at the test item concentrations of 2.7 and 32 mg/L based on mean measured concentrations (results of Dunnett’s tests, one-sided smaller, alpha = 0.05) after the test period of 72 hours. For the yield of the algae, a statistically significant inhibitory effect was also determined at the highest mean measured concentration of 96 mg/L.

However, the inhibition of the growth rate and yield was not estimated as a biologically relevant toxic effect, since the inhibition followed no dose-response-relationship and was <10% at the three highest test concentrations. Furthermore, in accordance with the test guidelines an effect within a range of 10 to 20% appears to be appropriate to represent the NOEC.

The 72‑hour NOEC was, therefore, determined to be higher than or equal to 96 mg/L (mean measured test concentration).
Based on nominal concentrations the NOEC is higher than or equal to 100 mg/L.

The EC10, EC20, and EC50 could not be calculated. The EC20 and EC50 were determined from the raw data to be >96 mg/L based on mean measured concentrations, and to be >100 mg/L based on nominal concentrations.
The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to this test item concentration.

In the control the biomass increased by a factor of 43 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 23%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled.
The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 0.5%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

At the start of the test, the pH measured in the treatments was between 7.9 and 8.0. At the end of the test, pH values of 8.1 were measured. The pH of the control medium did not increase by more than 1.5 units during the test. The water temperature during the test was maintained at 22 °C.
Results with reference substance (positive control):
The result of the latest positive control test performed in March 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.94 mg/L (study C86922), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71 1.7 mg/L).
Reported statistics and error estimates:
The Dunnett’s tests (one-sided smaller, alpha = 0.05) showed statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) at the test item concentrations of 2.7 and 32 mg/L, however these effects have been shown not to be related to biological toxic effects.

Any other information on results incl. tables

Table 1: Average Growth Rates (µ)

Nominal
test item concentration

(mg/L)

Mean measured concentration (geom.mean)

(mg/L)

Average growth rate µ (day-1) and inhibition of µ (Ir)

0-24 h

0-48 h

0-72 h

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

Control

---

1.21

0.0

1.38

0.0

1.25

0.0

1.0

0.54

1.14

6.3

1.35

2.2

1.26

-0.3

3.2

2.7

1.14

6.1

1.35

2.8

1.21*

3.7

10

9.7

1.08*

11.1

1.34

3.6

1.24

0.6

32

32

1.11

8.9

1.28*

7.3

1.22*

2.6

100

96

0.92*

24.1

1.15*

16.6

1.22

2.2

*:    mean value statistically significantly lower than in the control (according to Dunnett’s-tests, one-sided smaller,a= 0.05), however not estimated as a biologically relevant toxic effect

Table 2: Yield (Y)

Nominal
test item concentration

(mg/L)

Mean measured concentration (geom. mean)

(mg/L)

Yield Y (x 103) and inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

Y

Iy(%)

Y

Iy(%)

Y

Iy(%)

Control

---

7.1

0.0

44.8

0.0

124.9

0.0

1.0

0.54

6.4

10.2

42.2

5.8

126.5

-1.2

3.2

2.7

6.4

10.0

41.5

7.5

108.6*

13.1

10

9.7

5.9*

17.5

40.3

10.1

122.1

2.3

32

32

6.1

14.5

36.2*

19.3

113.0*

9.5

100

96

4.5*

36.1

27.2*

39.3

115.1*

7.9

*:    mean value statistically significantly lower than in the control (according to Dunnett’s-tests, one-sided smaller,a= 0.05), however not estimated as a biologically relevant toxic effect


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
This study is classified as Klimisch 1 and satisfies the guideline requirements for an acute toxicity study with algae.
Based on the results of this study, guetol would not be considered as harmful for algae.
Executive summary:

The influence of the test item GUETOL on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72‑hour static test (Harlan, 2010), according to OECD Guideline 201 (2006), the EU Commission Directive 92/69/EEC, C.3 (1992) and the Commission Regulation (EC) No 761/2009, C.3.

 

The nominal concentrations of the test item of 1.0, 3.2, 10, 32 and 100 mg/L were tested in parallel with a control.

 

The test was performed using Erlenmeyer flasks completely filled with test medium and tightly sealed with glass stoppers to avoid losses of test item (closed system).

 

The measured concentrations of the test item in the test media of the nominal test concentrations of 1.0 to 100 mg/L were between 98 and 102% of the nominal values at the start of the test. Thus, the correct dosing of the test item GUETOL was confirmed. During the test period of 72 hours, a decrease of test item concentration in the test media of the two lowest test item concentrations occurred. At the end of the test, 29 to 97% of the nominal values were found.

 

The biological results were related to the mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and at the end of the test).

 

The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) at the test item concentrations of 2.7 and 32 mg/L based on mean measured concentrations (results of Dunnett’s tests, one-sided smaller,  α =0.05) after the test period of 72 hours. For the yield of the algae, a statistically significant inhibitory effect was also determined at the highest mean measured concentration of 96 mg/L.

 

However, the inhibition of the growth rate and yield was not estimated as a biologically relevant toxic effect, since the inhibition followed no dose-response-relationship and was <10% at the three highest test concentrations. Furthermore, in accordance with the test guidelines an effect within a range of 10 to 20% appears to be appropriate to represent the NOEC.

The 72‑hour NOEC was, therefore, determined to be higher than or equal to 96 mg/L (mean measured test concentration).

Based on nominal concentrations the NOEC is higher than or equal to 100 mg/L.

The EC10, EC20, and EC50 could not be calculated. The EC20 and EC50 were determined from the raw data to be >96 mg/L based on mean measured concentrations, and to be >100 mg/L based on nominal concentrations.

Based on the results of this study, guetol would not be considered as harmful for algae.