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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
other: Analytical method developed for Daphnia and algae medium

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
july 2011
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
december 2015
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD guidance document No. 23, second edition, GUIDANCE DOCUMENT ON AQUEOUS-PHASE AQUATIC TOXICITY TESTING OF DIFFICULT TEST CHEMI-CALS
Version / remarks:
February 2019
Qualifier:
equivalent or similar to guideline
Guideline:
other: SANCO/3029/99: “Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414”
Version / remarks:
rev. 4 dated 11. July 2000
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,6-anhydro-β-D-glucose
EC Number:
207-855-0
EC Name:
1,6-anhydro-β-D-glucose
Cas Number:
498-07-7
Molecular formula:
C6H10O5
IUPAC Name:
(1R,2S,3S,4R,5R)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol
Constituent 2
Chemical structure
Reference substance name:
Glycollaldehyde
EC Number:
205-484-9
EC Name:
Glycollaldehyde
Cas Number:
141-46-8
Molecular formula:
C2H4O2
IUPAC Name:
2-hydroxyacetaldehyde
Constituent 3
Reference substance name:
Organic acids
IUPAC Name:
Organic acids
Constituent 4
Reference substance name:
Ketones
IUPAC Name:
Ketones
Constituent 5
Reference substance name:
Phenols
IUPAC Name:
Phenols
Constituent 6
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
Constituent 7
Reference substance name:
Oligomers of sugars and anhydrosugars
IUPAC Name:
Oligomers of sugars and anhydrosugars
Test material form:
liquid: viscous

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: At the end of the test, an extra replicate for each treatment and the control without algae was prepared for analytical verification purpose. To avoid adsorption onto the algae cells, the content of the TOC and the content of the two components of the test item in the test solutions was measured in the extra replicates without algae cells at the beginning and at the end of the experiment.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because the test item is a poorly water soluble UVCB substance with constituents of different water solubility, in accordance with the OECD guidance document no. 23, water-accommodated fractions (WAF) were prepared for the test.
- Loading: WAF was created by weighing the nominal load of 100 / 56 / 32 / 18 / 10 mg/L, adding the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 ± 1 hours. Hence, the real load was 100.3 / 55 / 32.8 / 17.8 / 10 mg/L
- Evidence of undissolved material: To remove undissolved compo-nents the solutions were filtrated through 0.45 µm PTFE filters
- Other relevant information: As the test item is highly viscous, it was placed in a water bath (30 ± 2 °C) and stirred thoroughly with a stirring staff until it is visibly homogeneous before usage.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Age of inoculum (at test initiation): cell concentration of 3 *103 cells/mL
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared
- pre-culture: For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 4 days under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 21– 24 °C). The resulting culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of the culture was determined.

Study design

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

Test conditions

Test temperature:
21.5 – 23.2 °C
pH:
7.3-7.9
Nominal and measured concentrations:
10 / 18 / 32 / 56 / 100 mg/L nominal concentration
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL
- Initial cells density: 3 *103 cells/mL
- Control end cells density: 571120 cells/mL
- volume solution: 45 ±1 mL
- No. of vessels per concentration (replicates): 3test solution+algae, 1 test solution only
- No. of vessels per control (replicates): 6 algae medium+algae, 1 algae medium only

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: under continuous lighting
- Light intensity and quality: within the specified range (4440 – 8880 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Other: appearance of the algae and detect abnormalities

TEST CONCENTRATIONS
- Range finding study
- Results used to determine the conditions for the definitive study: yes

CULTURING APPARATUS
-Details on culturing apparatus used: orbital shaker
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
23.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: concentrations refer to nominal loading rates
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
7.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: concentrations refer to nominal loading rates
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: concentrations refer to nominal loading rates
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
11.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
yield
Remarks on result:
other: concentrations refer to nominal loading rates
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
7.77 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
yield
Remarks on result:
other: concentrations refer to nominal loading rates
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
yield
Remarks on result:
other: concentrations refer to nominal loading rates
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
<= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
yield
Remarks on result:
other: concentrations refer to nominal loading rates
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
<= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: concentrations refer to nominal loading rates
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72h ErC50 0.64 mg/L (0.58 – 0.71 mg/L), 72h EyC50 0.32 mg/L (0.31 – 0.33 mg/L)
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software. Calculation was performed with Microsoft Excel® and ToxRat® Professional, version 3.3.0.

Any other information on results incl. tables

Not-GLP pre-test results (72h)

Nominal Concentration in mg/L

Inhibition in %

Growth rate

Yield

Blank control

0

0

1

0.8

4.7

10

2.9

15.4

100

60.6

97.0

Cell Number/mL

Nominal Concentration in mg/L

Parameter

Cell Number/mL

0 h

24 h

48 h

72 h

Blank control

Mean

3020

16470

107073

571120

SD

0

1941

5571

16190

10

Mean

3020

12273

63193

391460

SD

0

876

8181

12590

18

Mean

3020

8520

16447

48660

SD

0

1507

1052

2520

32

Mean

3020

7467

14153

27487

SD

0

429

866

853

56

Mean

3020

8020

17487

18580

SD

0

439

760

352

100

Mean

3020

14980

15380

14980

SD

0

886

661

629

Microscopical Observations

Nominal Concentration in mg/L

Normal and Healthy Appearance of the Algae

Blank control

Yes

10

Yes

18

No cells visible

32

No cells visible

56

No cells visible

100

No cells visible

Growth Rate µ, Yield

Nominal Concentration in mg/L

Parameter

Growth Rate (0-72 h) [day-1]

Yield (0-72 h)
[Cell Concentration/mL]

Blank control

Mean

1.75

568100

SD

0.01

16190

10

Mean

1.62

388440

SD

0.01

12590

18

Mean

0.93

45640

SD

0.02

2520

32

Mean

0.74

24467

SD

0.01

853

56

Mean

0.61

15560

SD

0.01

352

100

Mean

0.53

11960

SD

0.01

629

SD =Standard Deviation

Inhibition Values

Nominal concentration in mg/L

% Inhibition

Growth Rate (0-72 h)

Yield (0-72 h)

Blank control

0

0

10

7.2

31.6

18

47.0

92.0

32

57.9

95.7

56

65.3

97.3

100

69.5

97.9

Biological Results of the Test Item

Parameter

Value

95 % confidence interval

NOEL (Growth Rate) 72 h

< 10 mg/L

--

NOEL (Yield) 72 h

< 10 mg/L

--

LOEL (Growth Rate) 72 h

≤ 10 mg/L

--

LOEL (Yield) 72 h

≤ 10 mg/L

--

72h ErL10

7.96 mg/L

5.26 – 12.02 mg/L

72h EyL10

7.77 mg/L*

7.27 – 8.30 mg/L

72h ErL;50

23.96 mg/L

14.52 – 38.79 mg/L

72h EyL50

11.62 mg/L

10.74 – 12.53 mg/L

Analytical Determination, LC-MS-MS

A LC-MS-MS-Method for the determination of Pyrolytic Sugar was validated using two components as markers. At the beginning, the measured concentrations lay between 98 % and 106 % of the loading rates for component 1 and between 95 % and 100 % of the loading rates for component 2. At the end of the test, most likely due to an incorrect sampling which cannot be explained afterwards, the measured concentrations for both components at the concentration 18 mg/L and 32 mg/L were not plausible.

However, the treatments were prepared correctly. This was confirmed by the measured start concentrations of both components and the plausible measured TOC concentration at the beginning and at the end of the test as well as the plausible inhibition values in the algae test

In the other treatments the measured concentrations at the end of the test lay between 95 % and 119 % of the loading rates for component 1 and between 72 % and 79 % of the loading rates for component 2.

The details are given in the following tables:

Measured Concentrations and % of Nominal Loading, Component 1

Nominal Concentration in mg/L

Measured Concentration in mg/L

 t = 0 h

% of Nominal Loading

t = 0 h

Measured Concentration in mg/L

t = 72 h

% of Nominal Loading

t = 72 h

10

10.56

106%

9.53

95%

18

17.86

99%

< LOQ*

0%*

32

33.42

104%

11.65*

36%*

56

57.46

103%

59.37

106%

100

98.03

98%

119.44

119%

* implausible values, very likely caused by an error during sampling

Measured Concentrations and % of Nominal Loading, Component 2

Nominal Concentration in mg/L

Measured Concentration in mg/L

 t = 0 h

% of Nominal Loading

t = 0 h

Measured Concentration in mg/L

t = 72 h

% of Nominal Loading

t = 72 h

10

9.70

97%

7.20

72%

18

17.59

98%

0.26

1%*

32

31.98

100%

13.39

42%*

56

53.35

95%

44.45

79%

100

99.59

100%

79.29

79%

* implausible values, very likely caused by an error during sampling

Analytical Determination, TOC

Additionally, the total amount of dissolved test item in the test solutions was estimated by calculation based on the carbon content of the test item (48.73 %) and TOC measurement. The test item concentration based on TOC measurement lay between 91 % and 101 % of the loading rates at the beginning of the test, and between 74 % and 98 % at the end of the test.

The almost identical TOC concentrations in the respective test solutions at the beginning and end of the test confirms that the concentration of the test substance or the test sub-stance and its degradation products was stable over the test period.

Measured Concentrations IC and TC

Nominal
Concentration Test Item

Measured

TC
 t = 0 h

Measured

TC
 t = 72 h

Measured

IC
 t = 0 h

Measured

IC
 t = 72 h

mg/L

mg/L

mg/L

mg/L

mg/L

Blank control

7.41

7.43

7.77

7.55

10

12.42

11.84

7.51

7.44

18

15.49

14.05

7.51

7.55

32

21.91

20.30

7.33

6.96

56

31.75

30.12

6.77

5.91

100

52.85

52.65

6.78

4.75

Calculated Concentrations TOC

Nominal
Concentration Test Item

Calculated TOC (TC-IC)
 t = 0 h

Calculated TOC (TC-IC)
 t = 72 h

mg/L

mg/L

mg/L

Blank control

0

0

10

4,90

4,40

18

7,98

6,50

32

14,58

13,34

56

24,98

24,20

100

46,07

47,90

Calculated Test item Concentrations

Nominal
Concentration Test Item

Calculated

Concentration

 Test Item
 t = 0 h

Calculated

Concentration

 Test Item
 t = 72 h

% of Nominal concentration
t = 0 h

% of Nominal concentration
t = 72 h

mg/L

mg/L

mg/L

%

%

10

10.06

9.03

101%

90%

18

16.37

13.34

91%

74%

32

29.91

27.38

93%

86%

56

51.26

49.67

92%

89%

100

94.54

98.29

95%

98%

Within this study, 2 selected components of the UVCB were analytically verified. This to evaluate availability and compositional stability of the UVCB within the WAF. In addition, TOC was also monitored with the same purpose. 

This approach allowed confirmation of the presence of the substance in solution. However, due to variability of results and difficulty to link concentrations of components and/or TOC to overall nominal levels, the concentrations are presented as loading rates and not as re-calculated concentrations. This is in line with OECD Guideline 23 (difficult to test substances).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
72 h exposure of the green algae, Desmodesmus subspicatus, to Pyrolytic Sugar resulted in an EL50 growth of 23.96 mg/l and an EL50 yield of 11.62 mg/L
Executive summary:

One valid experiment was performed exposing for 72 hours the green algae, Desmodesmus subspicatu, to Pyrolytic Sugar according to OECD 201 guideline. Because the test item is a poorly soluble UVCB substance with constituents of different water solubility, in accordance with the OECD guidance document no. 23, water-accommodated fractions (WAF) were prepared for the test solution.

The study was performed using five loading rates 10 / 18 / 32 / 56 / 100 mg/L.

Analytical monitring was performed with a LC-MS-MS-Method validated using two components as markers, chosen because they could be clearly assigned as components of the test item and could be quantified in aqueous medium during validation. At the beginning, the measured concentrations lay between 98 % and 106 % of the loading rates for component 1 and between 95 % and 100 % of the loading rates for component 2. At the end of the test, most likely due to an incorrect sampling, the measured concentrations for both components at the concentration 18 mg/L and 32 mg/L were not plausible. However, the treatments were prepared correctly. This was confirmed by the measured start concentrations of both components and the plausible measured TOC concentration at the beginning and at the end of the test as well as the plausible inhibition values in the algae test.

In the other treatments the measured concentrations at the end of the test lay between 95 % and 119 % of the loading rates for component 1 and between 72 % and 79 % of the loading rates for component 2.

 

Additionally, the total amount of dissolved test item in the test solutions was estimated by calculation based on the carbon content of the test item (48.73 %) and TOC measurement. The test item concentration based on TOC measurement lay between 91 % and 101 % of the loading rates at the beginning of the test, and between 74 % and 98 % at the end of the test.

The almost identical TOC concentrationsand the measured concentrations of the both markersin the respective test solutions at the beginning and end of the test confirms that the concentration of the test substance or the test substance and its degradation products was present in the test solutions over the test period.

As the test item is a UVCB substance, according to OECD guidance document No. 23 (2019), the results are presented, based on the nominal WAF loading rates (see OECD guidance document no 23, § 177). Growth rate µ and the yield were determined from the cell number.

Significant inhibition of algal growth was observed at all tested loading rates for both endpoints (growth rate and yield).

At the two highest loading rates 56 mg/L and 100 mg/L, algae growth was almost completely inhibited. The three lowest loading rates showed a clear dose-response relationship.  

 

The following results for the test item, Pyrolytic Sugar, were determined after 72 hours exposure:

Endpoint

NOEL

LOEL

EL10

EL50

Growth Rate

< 10 mg/L

≤ 10 mg/L

7.96 mg/L

23.96 mg/L

Yield

< 10 mg/L

≤ 10 mg/L

7.77 mg/L*

11.62 mg/L

* extrapolated by the program

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