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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:
26 February 2007 - 02 March 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP. Study on ammonium salt.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC directive 92/69, Part C.3, 1992
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO International Standard 8692:2004
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 6675
- Substance type: single-constituent substance
- Physical state: clear colorless liquid
- Analytical purity: 29.9 ±0.1% in water
- Lot/batch no.: 140499-19/10
- Stability under test conditions: Stable in water at least 96 hours
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Control and test substance vessels had 2 mL aliquots taken at 0h, 24h, and 96h.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method: ADONA consists of 29.9% active ingredient (a.i.) in water. The lower concentrations were prepared by subsequent dilutions of the highest test concentration (334 mg/L in test medium). No special treatment other than a short magnetic stirring period was necessary to completely dissolve the test substance in the test medium.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None, final test solutions were all clear and colorless.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E+04 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: None
- Culturing media and conditions (same as test or not): cultures maintained under the same conditions as those used for testing.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
Combined limit/range-finding test

Test conditions

Hardness:
24 mg/L CaCO3
Test temperature:
22.1 - 22.5 °C
pH:
8.0 - 9.9
Nominal and measured concentrations:
Nominal concentrations: 0.334, 3.34, 33.4, and 334 mg/L
Corresponding to analytically confirmed active ingredient concentrations: 0.1, 1.0, 10, and 100 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-glass container, 50 mL fill volume
- Agitation: Yes, during incubation the algal cells were kept in suspension by continuous shaking
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 742.4E+04 cells/mL
- No. of vessels per concentration (replicates): 3 (each treatment group except the highest, with algae), 1 (each treatment group, without algae), 6 (highest concentration), 1 (each test group for sampling)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, M1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water purified by reverse osmosis; (Milli-RO water, Millipore Corp., Bedford, Mass., USA)
- Culture medium different from test medium: yes, propagation in M1, pre-culture established in M2 medium 3 days before test initiation to use in test, test in M2
- Intervals of water quality measurement: pH at beginning and end of test, temperature continuously

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous using TLD-lamps (cool-white)
- Light intensity and quality: 30 Watt, with a light intensity within the range of 78 - 84 µE/(m²∙s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):

- Determination of cell concentrations: Cell counts at 0h were determined using a microscope and a counting chamber. For 24h, 48h, and 72h samples, they were determined by spectrophotometric measurement at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength = 20 mm). Algal medium was used as a blank and the extra replicates as background for the treated solutions.
- Other: Appearance of cells were preformed at the end of the test by microscope to verify appearance of the inoculum culture and to observe for any abnormal appearance of the algae. TEST CONCENTRATIONS - Spacing factor for test concentrations: 1.8
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Exposure concentrations were based on nominal concentrations as analytical results showed that concentrations were stable and in agreement with nominal (with and without algae) within 99 -112% during the 96h test period.
Details on results:
- Exponential growth in the control (for algal test): yes/no: Yes (Figure 1)
- Growth in controls (validity criteria):
- cell density increase to hour 48: factor of >16
- % Coefficient of variance in average specific growth rate at hour 96: 1% (Table 1)
- % Coefficient of variance in section-specific growth rates to hour 96: ≤ 7% (Table 2)

Test concentrations and effect parameters can be found in Table 3 and Table 4, respectively.

- Any visual signs of phytotoxicity (abnormalities): None noted
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 72h ErC50: 2.0 mg/L, 95% C.I. 1.7 - 2.4 mg/L. The historical ranges for growth rate reduction for Pseudokirchneriella subcapitata lie between 0.82 and 2.3 mg/l. Hence, the growth rate reduction 72h ErC50 for the Pseudokirchneriella subcapitata tested corresponds with this range.
Reported statistics and error estimates:
Statistical analysis of the data was not needed as the effects recorded were not significant (<10%). No EC50-values could be calculated because the test substance proved to be non-toxic (EC50> maximum concentration test substance).

Any other information on results incl. tables

Table 1. Cell Density, Yield Inhibition, and Growth Rate Reductions

Nominal Concentration¹ ADONA

[mg/L]

Vessel Number

Individual Cell Densities

[104cells/mL]

Yield

Yield Inhibition [%]

Growth Rate [µ]

Growth Rate Reduction [%]

0h

24h

48h

72h

96h

0-96h

0-96h

0-96h

0-96h

1

1

7.31

44.00

240.41

757.16

756.16

0.06906

2

1

7.28

37.58

238.96

725.58

724.58

0.06861

3

1

7.38

37.74

255.30

797.60

796.60

0.06960

Control

4

1

8.35

47.80

278.67

782.14

781.14

0.06940

5

1

8.13

34.35

257.46

697.06

696.06

0.06820

6

1

8.68

45.02

247.45

694.98

693.98

0.06817

Mean

741.42

0.06884

CV

1%

1

1

9.85

44.38

249.78

681.44

680.44

8

0.06796

1

0.1

2

1

8.76

41.69

235.24

749.92

748.92

-1

0.06896

0

(0.334)

3

1

9.46

45.30

278.14

767.18

766.18

-3

0.06920

-1

Mean

731.85

1.3

0.06870

0.2

1

1

8.49

46.65

261.18

787.94

786.94

-6

0.06947

-1

1.0

2

1

8.05

47.84

251.57

762.86

761.86

-3

0.06914

0

(3.34)

3

1

8.08

41.22

243.02

697.46

696.46

6

0.06820

1

Mean

748.42

-0.9

0.06894

-0.1

1

1

8.00

62.89

248.56

680.30

679.30

8

0.06794

1

10

2

1

8.58

48.13

243.65

739.62

738.62

0

0.06881

0

(33.4)

3

1

7.84

47.65

264.82

723.10

722.10

3

0.06858

0

Mean

713.34

3.8

0.06845

0.6

1

1

8.04

49.17

252.29

740.90

739.90

0

0.06883

0

2

1

7.45

38.09

228.82

770.66

769.66

-4

0.06924

-1

3

1

7.89

38.38

225.05

659.48

658.48

11

0.06762

2

100

4

1

8.03

43.58

264.82

726.80

725.80

2

0.06863

0

(334)

5

1

8.12

45.44

251.22

803.98

802.98

-8

0.06968

-1

6

1

8.11

47.17

255.17

728.68

727.68

2

0.06866

0

Mean

737.42

0.5

0.06878

0.1

1) Analytically confirmed nominal concentrations of the active ingredient corrected for purity of the test product (29.9%). Nominal total product concentration in parentheses [mg/L].

Table 2 Growth rate: section-by-section

Nominal Concentration¹ ADONA

[mg/L]A

Vessel

Number

Growth rate (µ)

Growth rate reduction (%)

0-24h

24-48h

48-72h

72-96h

0-24h

24-48h

48-72h

72-96h

1

0.08289

0.07479

0.07076

0.04780

2

0.08271

0.06839

0.07708

0.04628

3

0.08328

0.06800

0.07965

0.04747

Control

4

0.08843

0.07270

0.07346

0.04300

5

0.08732

0.06004

0.08393

0.04150

6

0.09004

0.06859

0.07100

0.04303

Mean

0.08578

0.06875

0.07598

0.04485

CV%

4

7

7

6

Mean CV for section-by-section specific growth rate was 6%

1

0.09531

0.06272

0.07199

0.04182

-11

9

5

7

0.1

2

0.09042

0.06500

0.07210

0.04831

-5

5

5

-8

(0.334)

3

0.09363

0.06526

0.07562

0.04227

-9

5

0

6

Mean

0.09312

0.06433

0.07324

0.04413

-8.6

6.4

3.6

1.6

1

0.08912

0.07099

0.07177

0.04601

-4

-3

6

-3

1.0

2

0.08690

0.07426

0.06916

0.04622

-1

-8

9

-3

(3.34)

3

0.08706

0.06790

0.07393

0.04393

-1

1

3

2

Mean

0.08769

0.07105

0.07162

0.04539

-2.2

-3.3

5.7

-1.2

1

0.08664

0.08591

0.05726

0.04195

-1

-25

25

6

10

2

0.08956

0.07185

0.06758

0.04627

-4

-5

11

-3

(33.4)

3

0.08580

0.07519

0.07147

0.04185

0

-9

6

7

Mean

0.08733

0.07154

0.06543

0.04336

-1.8

-4.1

13.9

3.3

1

0.08685

0.07545

0.06814

0.04489

-1

-10

10

0

2

0.08368

0.06799

0.07471

0.05060

2

1

2

-13

100

3

0.08607

0.06591

0.07370

0.04480

0

4

3

0

(334)

4

0.08680

0.07048

0.07519

0.04207

-1

-3

1

6

5

0.08726

0.07175

0.07125

0.04847

-2

-4

6

-8

6

0.08721

0.07336

0.07034

0.04372

-2

-7

7

3

Mean

0.08631

0.07082

0.07222

0.04576

-0.6

-3.0

4.9

-2.0

1) Analytically confirmed concentrations of active ingredient, corrected for purity of the test product (29.9%). Nominal concentrations of total product presented in parentheses.

Table 3. Concentrations of ADONA in test medium

Time of sampling

(hours)

Concentration¹

Nominal (mg/L)

Measured (mg/L)

Relative to nominal (%)

Relative to initial (%)

0

n.d.²

n.a.³

0

99.9

99.1

99

99.9 (abiotic)

111

112

0

n.d.²

n.a.³

24

99.9

104

104

105

99.9 (abiotic)

109

109

98

0

n.d.²

n.a.³

96

99.9

104

104

105

99.9 (abiotic)

110

110

98

1) Concentrations for active ingredient, corrected for purity of test substance (29.9%)

2) Not detected

3) Not applicable

Limit of detection (LOD): 0.0056 mg/L, taking a dilution factor of 2 into account

Table 4. Effect parameters

Parameter

Concentration ADONA (mg/L)

Total Product

Active ingredient ¹

NOERC

334

100

96h-ERC50

> 334

> 100

NOEYC

334

100

96h-EYC50

> 334

> 100

1) Analytically confirmed nominal concentration of active ingredient corrected for purity of the test product (29.9%)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control cell densities increased by avg. factor >16 within 2 days. Coefficient of variation of sec.-by-sec. growth during whole test in controls <35%. Coefficient of variation of avg. specific growth during whole test in replicate controls <7%.
Conclusions:
The 96-hour ErC50 of ADONA to Pseudokirchneriella subcapitata is >100 mg/L (as active ingredient). The 96-hour NOEC (growth rate) of ADONA to Pseudokirchneriella subcapitata is 100 mg/L (as active ingredient).
Executive summary:

DONA is an acid with pKa ca.2 and will be entirely dissociated in aquatic systems. Aquatic toxicity testing would require neutralization of stock solutions, and DONA would in practice be testing of a neutral salt. This test examined the ammonium salt of DONA (ADONA).

The toxicity of ADONA to the green algae, Pseudokirchneriella subcapitata, was assessed in a 96-hour toxicity test conducted according to the OECD 201 (2006) method. An exponential growth was observed during the entire period of exposure in the control vessel and validity criteria were met. Exposure concentrations were based on nominal concentrations as analytical results showed that concentrations were stable and maintained within 99 -112% of nominal during the 96h test period.

The 96-hour ErC50 of ADONA to Pseudokirchneriella subcapitata for growth rate was > 100 mg/L (active ingredient).

The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice (GLP) standards. Therefore, use of this study for DONA is reliable with restrictions and the results are suitable for purposes of Risk Assessment, Classification & Labeling, and PBT Analysis.

Results Synopsis

Test Type: static (based on data obtained using OECD 201 methodology)

96-hr ErC50: >100 mg/L active ingredient

96-hr NOEC: 100 mg/L active ingredient

The ErC50 and NOEC results correspond to nominally 334 mg/L total product.