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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:
2012 - 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with minor deviations
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
ISO algal medium for the definitive test contained 0.08 mg/L of FeCl3.6H2O instead of 0.064 mg/L. The temperature of the incubator deviated by more than 2°C during the definitive test. These deviations have no effect on the validity of the study.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
During the definitive test, duplicate aliquots (ca 20 mL) were removed from each prepared concentration at the commencement of the test as 0 h samples. At 72h, following the removal of 5 mL aliquots for cell counts, the expired solutions from each treatment group were pooled and duplicate aliquots (ca 20 mL) were removed. No analysis was conducted during the range finding test.

Test solution concentrations were analysed under the validated Charles River Study No. 220649 (Analytical Procedure No. AP.20664.D2).
Vehicle:
no
Details on test solutions:
Range Finding Test
Test solutions were prepared by serial dilutions (1:10) of a primary stock solution of m/p DIOL with ISO algal medium. The primary stock solution (100 mg/L) was prepared by adding m/p DIOL (99.99 mg) and making up to volume with ISO algal medium in a glass volumetric flask (1000 mL). The flask and contents was then ultrasonicated for ca 10 mins to aid dissolution. The untreated control was prepared using ISO algal medium only.

Two vessels were prepared at each test/control concentration. Aliquots (100 ml) of each prepared test concentration (including ISO algal medium as control) were dispensed into the appropriate test vessels. Each vessel was inoculated with ca 1.04 x 104 cells P. subcapitata/mL.

Definitive Test
Test solutions were prepared by serial dilutions (1:1) of a primary stock solution of m/p DIOL with ISO algal medium. The primary stock solution (100 mg/L) was prepared by adding m/p DIOL (100.08 mg) and making up to volume with ISO algal medium in a glass volumetric flask (1000 mL). The flask and contents were then ultrasonicated for ca 10 mins to aid dissolution. The untreated control was prepared with using ISO algal medium only.

Three vessels were prepared at each test concentration and six vessels for the control group. Aliquots (100 ml) of each prepared test concentration (including ISO algal medium as control) were dispensed into the appropriate test vessels. Each vessel was inoculated with ca 1.0 x 104 cells P. subcapitata/mL.

An additional vessel was prepared for the 6.25 mg/L and 100 mg/L treatment groups for the purpose of comparative chemical analysis. These were each prepared with 100 mL aliquots of the appropriate test solution. These were not inoculated with P. subcapitata
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The freshwater green alga P. subcapitata (CCAP 278/4) was used for this study. Starter cultures were obtained from Culture Centre of Algae and Protozoa (CCAP), Oban, UK on 11 September 2012.

Transfers of P. subcapitata were made regularly into fresh ISO algal 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:
Not specified
Test temperature:
The maximum-minimum temperature of test solutions in the incubator was recorded at 24, 48 and 72 h. Temperature was measured using an RS Components digital maximum-minimum thermometer.

The temperature of the environmental area (22.2-24.4°C) was out with the range specified in the protocol during the definitive test.
pH:
The pH of the prepared ISO algal medium and test solution was measured in the definitive test, at test initiation (0 h) and in all control and test flasks at 72 h. All pH measurements were made using a Sentron Argus pH meter.

The pH of each test concentration (7.22-9.15) remained within protocol specification during the definitive test.
Dissolved oxygen:
Not relevant
Salinity:
Not relevant
Nominal and measured concentrations:
Range finding test, nominal concentrations of m/p DIOL : 0.1, 1.0, 10 and 100 mg/L plus a control.
Definitive test nominal m/p DIOL concentrations: 6.25, 12.5, 25, 50 and 100 mg/L, together with an untreated control ; corresponding mean measured (0 and 72 h) concentrations of m/p DIOL: 5.87, 12.21, 23.77, 47.04 and 96.58 mg/L, respectively (concentrations were maintained over the 72 h test period, with concentrations in the range 95% to 101% of the initial measured values)
Details on test conditions:
Test flasks: glass Erlenmeyer flasks (250 mL capacity) capped with foil lids, were used as the test flasks. Flasks were acid washed, rinsed in deionised water and dried at ca 80°C prior to use.

The test was conducted in a Sanyo Gallenkamp Orbital Incubator, set to maintain an environmental chamber temperature in the range 21-24°C. The flasks were provided with continuous uniform illumination in the spectral range 400 700 nm, obtained with cool white fluorescent tubes (Sylvania, Germany). Light intensity was measured at test initiation in 3 different areas of the incubator by a Lutron LX-101 lux meter. The flasks were rotated orbitally at ca 100 r.p.m. throughout the study, to facilitate the transfer of CO2 and maintain algal cells in suspension.

Algal cell concentrations were measured in the inoculum and in each flask at 72 h in the range finding test and at 24, 48 and 72 h in the definitive test. 5 mL aliquots were taken from each flask, at each timepoint and 100μl of Lugol Solution was added to each to preserve the sample for storage. Aliquots were stored within a fridge set to maintain ca 4°C until cell concentrations were determined. Cell concentration was determined using a compound light microscope and Improved Neubauer ruling counting chambers. Cell concentrations were determined within 7 days of sampling.


Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Range Finding Test
The results demonstrated that algal growth was inhibited by 13% at 100 mg/L and 12% at 10 mg/L. There was no significant inhibition evident at the 0.1 (3%) and 1 mg/L (5%) concentrations. The low percentage inhibition value observed in the 0.1 and 1 mg/L is likely to result from variation between the two replicates.

Definitive Test: See "Any other information on results incl. tables"
Chemical analysis of test samples taken at 0 and 72 h indicated that concentrations of m/p DIOL were maintained over the 72 h test period, with concentrations in the range 95 % to 101% of the initial measured values. Due to the confirmed stability in concentrations over the test period (overall mean measured concentrations of m/p DIOL did not deviate by greater than 20%), the following results are based on nominal concentrations. The overall mean measured concentrations of m/p DIOL were 5.87, 12.21, 23.77, 47.04 and 96.58 mg/L, for the nominal 6.25, 12.5, 25, 50 and 100 mg/L solutions, respectively. No m/p DIOL was detected during analysis of 0 h and 72h control samples.

There was no significant difference in measured concentrations of m/p DIOL between cell-free medium and medium containing cells, as indicated by the analysis of samples without algae at 72 h. This indicates no absorption/accumulation of m/p DIOL to/in algal cells (Table 2).

Algal cell concentrations recorded after 24, 48 and 72 h are presented in Table 3. Cell counts at 0, 24, 48 and 72 h were converted to dry weight equivalent (μg) by multiplying by 2.5. The average specific growth rate (μave) and yield were estimated for each replicate flask during the experimental period, using dry weight equivalents. The results are reported as mean cell concentrations (x105 cells/mL), μave/day, yield and growth rate and yield inhibition against the control.

Measured Concentrations of m/p DIOL During the Definitive Test

 

Nominal Concentration of m/p DIOL (mg/L)

Time (h)

Measured Concentrations (mg/L)

Mean Measured Concentrations (mg/L)

Deviation of 72 h Mean Measured Concentration from 0 h

(±%)

Deviation of Mean Measured Concentration from nominal (±%)

Control

0

ND

ND

NA

NA

72

ND

6.25

0

5.84

5.87

1

-6

72

5.89

72*

5.75 *

5.80 *

NA

NA

12.5

0

12.54

12.21

-5

-2

72

11.87

25

0

23.91

23.77

-1

-5

72

23.62

50

0

47.41

47.04

-2

-6

72

46.66

100

0

97.09

96.58

-1

-3

72

96.06

72*

95.76 *

96.43 *

NA

NA

* Flask not inoculated with algae

ND = Not Detected

NA = Not Applicable

 

Cell Growth Group Mean Values Determined from Daily Cell Counts During the Definitive Test

 

Growth Function

Time Interval

(h)

Nominal Concentration of m/p DIOL

(mg/L)

Control

6.25

12.5

25

50

100

Mean Cell Concentration (x105 cells/mL)

0-24

0-48

0-72

0.71

4.43

21.05

0.99

4.02

23.67

0.70

4.50

24.19

0.74

4.00

20.34

0.63

4.81

20.96

0.74

3.71

21.82

Average Specific Growth Rate (µave/ day)

0-24

0-48

0-72

24-48

48-72

1.95

1.89

1.78

1.83

1.56

2.28

1.84

1.82

1.41

1.78

1.95

1.90

1.83

1.85

1.68

1.98

1.84

1.77

1.71

1.62

1.83

1.94

1.78

2.04

1.47

1.99

1.81

1.79

1.62

1.77

Yield

0-24

0-48

0-72

1.52

10.82

52.39

2.22

9.79

58.93

1.51

11.01

60.22

1.59

9.74

50.61

1.32

11.78

52.15

1.59

9.03

54.30

 

pH values measured for Test Solutions at 0 h and for Replicate Flasks at 72 h During the Definitive Test

 

Nominal Concentration of m/p DIOL

(mg/L)

Replicate Flask

pH Value

0 h *

72 h

Control

I

7.72

9.15

II

9.05

III

9.12

IV

7.91

V

8.22

VI

8.04

6.25

I

7.73

8.16

II

7.86

III

8.96

A

7.59

12.5

I

7.54

7.22

II

7.57

III

8.74

25

I

7.61

7.39

II

7.68

III

8.99

50

I

7.57

7.52

II

9.03

III

8.94

100

I

7.60

7.64

II

7.48

III

7.88

A

7.54

* pH value recorded at 0h taken from stock solutions

A = Not inoculated algae flasks

 

 

Temperatureof Test Solutions Throughout the Definitive Test

 

Time (h)

Temperature (°C)

Maximum

Minimum

0-24

22.2

23.2

24-48

22.3

24.4

48-72

23.0

23.5

 

 

The average light intensity measured during the range finding and definitive test was 6600 and 8693 lux, respectively.


 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
The 72h EC50 for average specific growth rate and yield could not be calculated due to the lack of fractional inhibition data and it is therefore estimated to be >100 mg/L. The NOEC was estimated to be 100 mg/L.
Executive summary:

This study was designed to determine the effects of the reaction mass α, α, α', α'-tetramethyl-m-xylene-α, α'-diol and α, α, α', α'-tetramethyl-p-xylene-α, α'-diol (m/p DIOL) 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 results from the range finding test, the definitive test was conducted with nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L m/p DIOL, together with an untreated control. The corresponding mean measured (0 and 72 h) concentrations of m/p DIOL were 5.87, 12.21, 23.77, 47.04 and 96.58 mg/L, respectively. Chemical analysis of test samples taken at 0 and 72 h indicated that concentrations of m/p DIOL were maintained over the 72 h test period, with concentrations in the range 95% to 101% of the initial measured values.

 

The 72 h EC50 for average specific growth rate and yield could not be calculated due to the lack of fractional inhibition, and it is therefore estimated to be > 100 mg/L. The NOEC was estimated to be 100 mg/L.

OECD Guideline 201 validity criteria were met and therefore the test was considered to be acceptable.

 

Description of key information

The 72h EC50 for average specific growth rate and yield could not be calculated due to the lack of fractional inhibition data and it is therefore estimated to be >100 mg/L.  The NOEC was estimated to be 100 mg/L.

Key value for chemical safety assessment

Additional information