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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
For the purpose of chemical analysis during the definitive test, duplicate aliquots (ca 20 mL) were removed from test and control solutions at the commencement of the test as 0 h samples. At test termination, an aliquot (ca 20 mL) was removed from each replicate flask and combined separately, for the test and control solutions. Duplicate aliquots (ca 20 mL) were removed from the combined solutions as 72 h samples.
An additional flask was prepared at nominal loading rates of 4 and 0.25 mg benzyltoluene/L and these incubated (without algal cells) with the test and control flasks. At 72 h, duplicate aliquots (ca 20 mL) were removed from these additional flasks for analysis, to determine any differences in measured concentrations of benzyltoluene between cell-free medium and medium containing cells.
Vehicle:
no
Details on test solutions:
Individual WAF’s were prepared by adding weighed amounts of benzyltoluene (5.9, 11.6, 23.0, 46.0 and 92.1 mg) to algal medium (23 litre) and stirring these in the dark for ca 25 h. A control WAF (algal medium only) was prepared in an identical manner to that of the test WAF’s. The rate of stirring was adjusted to ensure the vortex was =1% of the depth of the water column. Following the period of stirring, the contents were allowed to settle for ca 25 h, after which ca 1100 mL of solution was siphoned from the centre of the vessels, with the first ca 100 mL being discarded. The remaining ca 1000 mL of solution was used as the test and control solutions and as samples for chemical analysis.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The freshwater green alga Pseudokirchneriella subcapitata CCAP 278/4 was used for this study. Starter cultures were obtained from CCAP Oban, UK on 14 July 2009 (limit test) and 09 March 2010 (definitive test).

Transfers of P. subcapitata were made regularly into fresh growth medium to provide suitable subcultures, in the exponential phase of growth, for test inoculations.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
All testing and algal culturing was conducted using ISO freshwater algal medium
Test temperature:
23.0- 23.4 °C
pH:
Initial 7.53
Final 8.44 - 8.96
Dissolved oxygen:
Not a parameter to be monitored in application of the Guideline
Salinity:
All testing and algal culturing was conducted using ISO freshwater algal medium
Nominal and measured concentrations:
Test solutions were prepared through the WAF method.
Loading rates were: 0 - 0.25 - 0.5 - 1 - 2 and 4 mg/L
Measured Initial concentrations were 0 - 212 - 281 - 306 - 414 - 505 µg/L
All final (72 h) measured concentrations were below limit of quantification (61 µg/L)
Concentrations used for calculation were geometric mean between initial and loq concentrations: 0 - 114 - 131 - 137 - 159 and 176 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: erlenmeyer flasks
- Material, size, headspace, fill volume: glass, 250 mL capacity, 100 mL medium volume
- Aeration: capped with foil lids
- Initial cells density:0.86 e+4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to ISO, as recoomended in OECD 201

OTHER TEST CONDITIONS
- Sterile test conditions: no
- adjustement of pH: no
- Photoperiod: continuous
- Light intensity and quality: uniform cool white, 8067 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: limit test with WAF loading rate 5 mg/L

Reference substance (positive control):
no
Key result
Duration:
3 d
Basis for effect:
growth rate
Remarks on result:
other: No adverse effect up to solubility limit
Details on results:
The following results are based on geometric mean measured concentrations of benzyltoluene:
From growth rate data, the EC10 at 24 h was estimated as 147 µg/L with 95% confidence limits of 128 and 169 µg/L. Due to the lack of growth inhibition, no other EC values could be estimated at any time point. It is concluded that benzyltoluene does not affect algal growth rate over a 72 h period, at the maximum achievable solubility under the conditions of the test.

The pH of test solutions and the temperature of the environmental area were maintained within protocol specification throughout the test. The average light intensity measured at flask level during the definitive test was 8067 lux. 
Reported statistics and error estimates:
Growth rates were analysed for homogeneity of variance using Levene's test at a 1% significance level. Following ANOVA, benzyltoluene treated groups were compared to the control using a one-sided Dunnett's test (tested for a decrease) at a 5% significance level. For regression analysis, a probit transformation was applied followed by regression against logarithmetically transformed concentrations of test material with a Newton-Raphson maximum likelihood iterative procedure being used to obtain parameter estimates. From the fitted model, the ECx value was estimated.

Geometric mean measured concentration of benzyltoluene (µg/L)

Replicate flask

Mean cell concentrations (x105cells/mL)

0 h (inoculum)

24 h

48 h

72 h

0 (control)

1

0.09

0.65

4.35

15.55

2

0.09

0.58

4.90

15.28

3

0.09

0.65

3.68

13.25

4

0.09

0.65

4.00

15.53

5

0.09

0.43

3.83

15.18

6

0.09

0.85

3.88

15.85

114

1

0.09

0.63

2.45

10.90

2

0.09

0.48

1.80

8.98

3

0.09

0.50

4.03

12.53

131

1

0.09

0.60

3.58

15.35

2

0.09

0.58

4.53

16.70

3

0.09

0.55

4.08

15.93

137

1

0.09

0.63

2.30

10.40

2

0.09

0.55

2.68

12.80

3

0.09

0.45

1.83

9.40

159

1

0.09

0.48

2.13

9.60

2

0.09

0.58

3.68

14.73

3

0.09

0.53

4.00

16.03

176

1

0.09

0.38

1.80

8.85

2

0.09

0.60

3.23

11.83

3

0.09

0.33

3.33

12.68

Table 1: Cell concentration per replicate per sampling time.

Geometric mean measured concentration of benzyltoluene (µg/L)

Time (h)

Measured concentrations of benzyltoluene (µg/L)

Geometric mean measured concentrations of benzyltoluene (µg/L)

Control

0

< LOD

0

72

< LOD

114

0

212

114

72

< LOD

131

0

281

131

72

< LOD

137

0

306

137

72

< LOD

159

0

414

159

72

< LOD

176

0

505

176

72

< LOD

 Table 2:Measured concentrations of benzyltoluene in test solutions during the test. LOD = 61 µg/L

 

Geometric mean measured concentration of benzyltoluene (µg/L)

Replicate flask

Mean cell concentrations (x105cells/mL)

0 h

72 h

0 (control)

1

7.53

8.84

2

8.93

3

8.90

4

8.91

5

8.93

6

8.81

114

1

7.53

8.73

2

8.44

3

8.92

131

1

7.52

8.96

2

8.98

3

8.87

137

1

7.53

8.90

2

8.93

3

8.62

159

1

7.53

8.88

2

8.82

3

8.95

176

1

7.51

8.67

2

8.92

3

8.93

 Table 3: pH evolution from 0h to 72h in each replicate during the test.

Validity criteria fulfilled:
yes
Conclusions:
The 72 h EC50 value for average specific growth rate and yield was estimated to be greater than the maximum solubility of benzyltoluene under the conditions of the test. It is concluded that benzyltoluene does not affect algal growth rate over a 72 h period, at the maximum achievable solubility under the conditions of the test.
Executive summary:

This study was designed to determine the effects of benzyltoluene on the growth of Pseudokirchneriella subcapitata, a unicellular freshwater green alga, over a 72 h period. The study was performed to meet the requirements of OECD (2006) Guideline 201.

Based on the outcome of a limit test, a definitive test was conducted with test solutions prepared as water accommodated fractions (WAF) from initial loading rates of 0.25, 0.5, 1, 2 and 4 mg benzyltoluene/L. A control of untreated algal medium was included in the test. The method of preparation was selected to ensure the test item was tested at the maximum achievable solubility, under the conditions of the test. 

Analytical samples taken at 0 h indicated that benzyltoluene is only partially soluble in algal medium, with concentrations ranging from 212 µg/L to 505 µg/L. Measured concentrations at 72 h indicated that the initial concentrations of benzyltoluene were not maintained. The loss over the 72 h period was ca100% with all test concentrations being below the limit of detection (limit of quantification = 61 µg/L). The overall (0 and 72 h) geometric mean measured concentrations of benzyltoluene ranged from 114 µg/L to 176 µg/L.

The 72 h EC50 and 72h EC10 values for average specific growth rate and yield were estimated to be greater than the maximum solubility of benzyltoluene under the conditions of the test. From the growth rate results of this study, it was not possible to derive a LOEC and thus it was not relevant to estimate a NOEC.

Therefore, it is concluded that benzyltoluene does not affect algal growth rate over a 72 h period, at the maximum achievable solubility under the conditions of the test.

ed to be greater than the maxim

Description of key information

In an OECD 201 TG compliant study performed under GLP, benzyltoluene did not affect algal growth rate over a 72 h period at the maximum achievable solubility.

Key value for chemical safety assessment

Additional information