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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:
24 Aug. - 03 Sep. 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Naphthalene oil
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Substance type: organic
- Physical state: solid
- Stability under test conditions: WAF (water accommodated fraction) limited
- Storage condition of test material: room temperature, exclusion of light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for TOC-analysis were taken at 0 h and 72 h (TOC-Analyser (DIMA-TOC 100).
Measurements of the cell concentrations per mL: at time 24 h, 48 h and 72 h by optical densitometry at 525 nm

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fractions (WAFs) prepared by stirring various amounts of the test item in Alga-medium
for 24 h with magnetic stirrers (open conditions). The WAFs were prepared in 2-L glass beakers.
The beakers were equipped with a glass tube that allowed the separation of the water phase by siphoning.
The test loading rates of Naphthalene Oil were weighed on microscope slides that afterwards were placed into the glass beakers.
The beakers were then filled with Alga-medium.
Mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction
of the test item. To ensure this, the vortex developed at the surface by stirring was set at ~ 10 % of the water depth.
After stirring for 24 h the WAFs were allowed to stand for 1 h before use to facilitate phase separation.
The extracts gained with this method were clear.

- Pre-conditioning: The test vessels of the Definite Alga-Test were rinsed with control media and WAFs for ca. 0.5 h before the definite
start of the tests to facilitate adsorption of the test item to the glass walls of the beakers.
Control media and WAFs were then discarded, and the test vessels were refilled again with control media and WAFs, respectively.
- Differential loading: control, 100 mg/L
- Controls: without test material (blank; Alga medium)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular green alga
- Strain: : SAG 86.81
- Source: Albrecht von Haller Institut für Pflanzenwissenschaften der Universität Göttingen

- Age of inoculum (at test initiation): ca. 72 h
[Pre-culture: Incubation in 250 mL Erlenmeyer flasks at ± 25 °C on a shaker
(80 - 120 rpm), ca. 72 h, ca. 10000 lux (Gro-Lux fluorescent tubes), permanent illumination]

- Method of cultivation: Sterile on agar tubes (OECD-Medium, 0.8 % Agar), transferred to a new agar tube every 2 months


Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
none

Test conditions

Hardness:
no data specified: Compositions of stock solutions given.
Test temperature:
24.3 - 24.8 °C (25 +-2°C)
pH:
initial (with algae): 8.0
final (with algae): 8.6 (control); 8.5 (100 mg/L)
Salinity:
no data specified: Compositions of stock solutions given.
Nominal and measured concentrations:
Time [h] Control 100 mg/L
=====================================
0 0.6 9.3 mg C/mL (TOC of WAFs)
72 2.8 5.6 mg C/mL (TOC of WAFs)
=======================================
The oil-related TOC was about 3 - 8 mg C/L for 100 mg oil/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL-Erlenmeyer
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- Initial cells density: approx. 5000 cells/mL
- Control end cells density: approx. 245600 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: permanent
- Light intensity and quality: approx. 10000 Lux (Gro-Lux fluorescent tubes)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: densitometrically at 525 nm (turbidity)
- Chlorophyll measurement: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Range finding study
- Results used to determine the conditions for the definitive study: based on results from daphnia test

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: slight from 48 - 72 h
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

- Observation of abnormalities (for algal test): no data

Reported statistics and error estimates:
Results of the measurements of the cell concentrations (cells per mL) were treated statistically according to Grubbs Test. Calculations of the growth curves, average specific growth rate and yield were accomplished taking into account the results of Grubbs Test.

Any other information on results incl. tables

Growth rates are listed below, from 0 -24, 24 - 48, and 48 - 72 h (Table 1) and average from 0 - 72 h (Table 2):

Table 1: Loading rates of the Test item, cells/mL in the test flasks at 24, 48 and 72 h with mean value,

section-by-section average specific growth rate [μ] during observation times 0-24 h, 24-48 h

and 48-72 h, mean value of the average specific growth rate [μ], standard deviation and

coefficient of variation Vc [%], inhibition [%] and mean value of inhibition of the average

specific growth rate [%] during observation times 0-24 h, 24-48 h and 48-72 h (Rep. Tab. 3)

[Note: Negative inhibition means increased growth, apparently stimulation]

Loading rate

[mg/L]

Cells / mL

Cells / mL

Mean value

Average Specific

growth rate

[µ]

Average Specific

growth rate

[µ]

Mean value

Standard deviation

Coefficient of variation Vc [%]

Inhibition of Average Specific
growth rate

[%]

Inhibition of Average Specific

growth rate

Mean value

[%]

24 h

Control 1

18000

20000

1.28

1.34

0.35

25.9

Control 2

29500

1.77

Control 3

16000

1.16

Control 4

11000

0.79

Control 5

21000

1.44

Control 6

24500

1.59

100

21000

20667

1.44

1.42

0.10

6.73

-7.21

-5.73

100

21000

1.44

-7.21

100

18000

1.28

4.31

100

19500

1.36

-1.67

100

24000

1.57

-17.2

100

20500

1.41

-5.40

Loading rate

[mg/L]

Cells / mL

Cells / mL

Mean value

Average Specific

growth rate

[µ]

Average Specific

growth rate

[µ]

Mean value

Standard deviation

Coefficient of variation Vc [%]

Inhibition of Average Specific
growth rate

[%]

Inhibition of Average Specific

growth rate

Mean value

[%]

48 h

Control 1

78500

100083

1.47

1.61

0.16

9.99

Control 2

116500

1.37

Control 3

95000

1.78

Control 4

55500

1.62

Control 5

114500

1.70

Control 6

140500

1.75

100

68500

80417

1.18

1.34

0.25

18.6

26. 8

16.8

100

61000

1.07

34.0

100

76500

1.45

10.4

100

94500

1.58

2.27

100

74500

1.13

29.9

100

107500

1.66

-2.62

Loading rate

[mg/L]

Cells / mL

Cells / mL

Mean value

Average Specific

growth rate

[µ]

Average Specific

growth rate

[µ]

Mean value

Standard deviation

Coefficient of variation Vc [%]

Inhibition of Average Specific
growth rate

[%]

Inhibition of Average Specific

growth rate

Mean value

[%]

72 h

Control 1

211500

245583

0.99

0.94

0.24

25.7

Control 2

248500

0.76

Control 3

244500

0.95

Control 4

220000

1.38

Control 5

261000

0.82

Control 6

288000

0.72

100

213500

231167

1.14

1.06

0.17

16.2

-21.5

-13.6

100

209500

1.23

-31.9

100

175500

0.83

11.2

100

283500

1.10

-17.4

100

249000

1.21

-29.0

100

256000

0.87

7.25

Table 2: Loading rate of the Test item, cells/mL in the test flasks at 72 h, average specific growth

rate [μ] over the whole testing period 0-72 h, mean value of the average specific growth

rate [μ], standard deviation and coefficient of variation Vc [%], inhibition [%] and mean value

of inhibition [%] of the average specific growth rate [μ] (Rep. Tab. 4)

Loading rate

[mg/L]

Cells / mL

Average

Specific

growth rate

[µ]

Average

Specific

growth rate

[µ]

Mean value

Standard deviation

Coefficient of variation

Vc [%]

Inhibition of Average

Specific

growth rate

[%]

Inhibition of Average

Specific

growth rate

Mean value

[%]

72 h

Control 1

211500

1.25

1.30

0.04

2.90

Control 2

248500

1.30

Control 3

244500

1.30

Control 4

220000

1.26

Control 5

261000

1.32

Control 6

288000

1.35

100

213500

1.25

1.27

0.04

2.83

3.46

1.73

100

209500

1.25

3.95

100

175500

1.19

8.50

100

283500

1.35

-3.83

100

249000

1.30

-0.49

100

256000

1.31

-1.20

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
1) Biomass in controls increased 49.1x in 72 h (nom. >= 16x). 2) Mean var-coeff. for section-spec. growth rates: 33.5 % (nom. ≤ 35%). 3) Var-coeff. of av. spec. growth rates for the whole period in replicate controls: 2.9% (nom. value ≤ 7%).