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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:
2009-07-21 to 2009-08-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, All validity criteria fulfilled, idenfication of test substance complete, including chemical analyses
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: From the series of concentrations the second lowest and the second highest test concentration (1.25 and 5.00 mg/L) and the control were analytically verified at the beginning of the test.
- Sampling method: Separate replicates for the test item analysis after 0 and 72 h were prepared without algae. Analysis after 72 h of these concentrations was carried out to demonstrate stability of the test item. Sorption to the walls of the glass container was checked out of one test replicate (prepared with algae) of a concentration level close to the EC50-value (the second lowest test concentration of 1.25 mg/L was chosen). Additionally, the freshly prepared stock solution of 10 mg/L was analytically verified to check their correct preparation.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshyl prepared with test medium. Dispersion treatment ultrasonification for 15 min at 40 °C and an equilibration phase for at least 1 h.
- Eluate: Natural river water
- Differential loading: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L
- Controls: Six replicates (without test item) were testedt under the same test conditions as the test replicates
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): -
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
After 72 h of exposure, 0.5 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from the test concentrations 1.25 – 10.0 mg/L and the control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 5 - 7 days under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 10.0 mg/L.
Recovery of Alga Cells

Nominal test item concentration [mg/L] Incubation time [d] Cell density [cells/mL] Growth rate after incubation time [d-1]
Initial# Final
10.0 7 86 277 0.17
216 0.13
433 0.23
5.00 215 2266 0.34
1200 0.25
699 0.17
2.50 5 10331 157738 0.55
159127 0.55
144564 0.53
1.25 27709 504934 0.58
343160 0.50
312771 0.48
Control 69362 1955932 0.67
1997801 0.67
Hardness:
Not measured
Test temperature:
Mean: 23 °C, Min: 22 °C, Max: 24 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
10.0 8.05 8.17
5.00 7.98 8.12
2.50 7.95 8.01
1.25 7.94 8.11
0.625 7.93 8.19
Control 7.93 8.05
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Concentrations and Recovery Rates of Tallowtripropylenetetramine
(CAS No. 85632-63-9)

Sampling date 200-07-21
Test start 2009-07-24
Test end (with algae) 2009-07-24
Test end (without algae)
Start of analysis 2009-07-24 2009-07-24 2009-07-24
Nominal concentration of test item
[mg/L] Tallowtripropylenetetramine
Meas.
conc.
[mg/L] RR

[%] Meas.
conc.
[mg/L] RR

[%] Meas.
conc.
[mg/L] RR

[%]
1.25 1.05 84 82.0 7 89.3 7
5.00 4.63 93 -- -- 462 9
( Absorption on the glass wall )
1.25 -- -- 168 13 -- --
Control < LOQ -- --
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 1387239 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: Yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water of the river Innerste was used. This river is located in
Sarstedt / Ruthe. Water parameters are given in Table 1. Additionally 50 % of the components concentrations of the dilution water (total application volume 6.5 mL/L) acc. to the guideline were added to enable a sufficient growth of algae
Characterisation Data of Natural River Water

River Innerste
Location D-31157 Sarstedt / Ruthe, Ruther Straße, Innerste
Sampling Date 2009-02-12
Weather on Day of Sampling dry, sunny, ca. 1 °C
Colour Slightly, turbid, yellowish
pH 8.08
Conductivity [µS/cm] 887
DOC [mg C/L] 2.8
TOC [mg C/L] 2.9
Ammonium-N [mg N/L] 0.163
Nitrate-N [mg N/L] 6.10
o-Phosphate-P [mg P/L] 0.103
Total Phosphate [mg P/L] 0.152
Suspended Matter [mg/L] 24
Total Hardness [mg CO3/L] 338




OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 h)

Nominal
Test Item Concentration [mg/L] Rate-related inhibition
[%] Yield Inhibition

[%]
10.0 100 100
1.00 1 3
0.10 -1 -7
0.01 -1 -3

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.27
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.999-1.45
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.47 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 2.23 - 2.71
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: (1.04-1.18)
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: 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
- EC50:
Rate-related inhibition: 0.695 (CI 0.657 - 0.729) mg/L
Yield Inhibition: 0.320 (CI 0.294 - 0.358) mg/L
Reported statistics and error estimates:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

EC10-, EC20-and EC50- values and 95 % Confidence Intervals of
Tallowtripropylenetetramine (CAS No. 68911-79-5) (0-72 h)

                                based on nominal test item concentrations [mg/L]

                               

Rate-related inhibition

ErC10

1.27 (0.999 - 1.45)

ErC20

1.60 (1.43 - 1.79)

ErC50

2.47 (2.23 - 2.71)

Inhibition of yield

EyC10

0.822 (0.758 - 0.894)

EyC20

0.881 (0.804 - 0.955)

EyC50

1.12 (1.04 - 1.18)

Cell Densities

 

Nominal
test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

10

1

5074

4135

11

408

 

2

5074

5254

0

0

 

3

5074

5808

1564

2163

 

Mean

5074

5066

525

857

 

5.00

1

5074

9518

2372

1900

 

2

5074

11212

2803

1900

 

3

5074

12454

2881

2660

 

Mean

5074

11061

2685

2153

 

2.50

1

5074

20818

37280

86639

 

2

5074

25107

52450

100608

 

3

5074

26341

39022

122695

 

Mean

5074

24089

42917

103314

 

1.25

1

5074

32991

172055

450482

 

2

5074

33467

220775

574716

 

3

5074

39432

224190

637360

 

Mean

5074

35297

205673

554186

 

0.625

1

5074

33184

530091

1807422

 

2

5074

35344

497144

1876658

 

3

5074

38497

552330

1812051

 

Mean

5074

35675

526522

1832044

 

Control

1

5074

52265

255137

1435322

 

2

5074

41937

278372

1610441

 

3

5074

42962

330032

1388623

 

4

5074

50244

226499

1208375

 

5

5074

42626

248237

1382994

 

6

5074

38551

241829

1297681

 

Mean

5074

44764

263351

1387239

 

Evaluation after 72 h

             

Nominal
test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

 10

1

-0.840

100.00

-4666

100.00

2

n.a.

100.00

-5074

100.00

3

-0.284

100.00

-2911

100.00

Mean

n.a.

100.00

-4217

100.00

5.00

1

-0.327

100.00

-3174

100.00

2

-0.327

100.00

-3174

100.00

3

-0.215

100.00

-2414

100.00

Mean

-0.290

100.00

-2921

100.00

2.50

1

0.946

49.39

81565

94.10

2

0.996

46.73

95534

93.09

3

1.062

43.19

117621

91.49

Mean

1.001

46.44

98240

92.89

1.25

1

1.495

19.99

445408

67.77

2

1.577

15.65

569642

58.79

3

1.611

13.80

632286

54.25

Mean

1.561

16.48

549112

60.27

0.625

1

1.959

-4.79

1802348

-30.40

2

1.971

-5.46

1871584

-35.41

3

1.959

-4.83

1806977

-30.74

Mean

1.963

-5.03

1826970

-32.18

Control

1

1.882

1430248

2

1.920

1605367

3

1.871

1383549

4

1.824

1203301

5

1.869

1377920

6

1.848

1292607

Mean

1.869

1382165

Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

VC %

Mean

VC %

section-by-section                           

0 - 24 h

24 - 48 h

48 - 72 h

Control

1

2.332

1.585

1.727

1.882

0.3966

21.1

15.8

2

2.112

1.893

1.755

1.920

0.1799

9.37

3

2.136

2.039

1.437

1.871

0.3788

20.2

4

2.293

1.506

1.674

1.824

0.4143

22.7

5

2.128

1.762

1.718

1.869

0.2254

12.1

6

2.028

1.836

1.680

1.848

0.1742

9.42

Mean

1.869

 

SD ±

0.03

 

VC %

1.73

 

Validity criteria fulfilled:
yes
Conclusions:
Tallowtripropylenetetramine (CAS No. 68911-79-5) was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal concentrations): The EC10-values with 95 % confidence intervals for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 1.27 (0.999 – 1.45) and 0.822 (0.758 – 0.894) mg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 2.47 (2.23 – 2.71) and 1.12 (1.04 – 1.18) mg/L, respectively.
The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 5 - 7 days of growth under test conditions.
The concentrations of Tallowtripropylenetetraminewere analysed at the concentration levels 1.25 and 5.00 mg/L (prepared without algae) and the control at test start and test end via LC-MS/MS analysis. The measured concentrations at test start were in the range of 84 – 93 % of the nominal values. At the end of the test Tallowtripropylenetetraminewas additionally analysed  at  the  concentration  level 1.25 mg/l (prepared  with algae) and  gave  recoveries of
7 - 9 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. Minor adsorption (13 % of nominal) of the test item to glassware was observed. Therefore all effect values are given based on nominal concentrations of Tallowtripropylenetetramine (CAS No. 68911-79-5).
Executive summary:

The toxicity of Tallowtripropylenetetramine (CAS No. 68911-79-5) (Batch no.: S001255)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from July 21 to August 02, 2009. The aim of the study was the determination of EC10 - , EC20 - and EC50 - values of growth rate and yield over a period of 72 h.

An individual test design was applied. Natural river water was used as dilution medium (for details and initial considerations please refer to page 12).

Tallowtripropylenetetramine(CAS no. 68911-79-5) is practically insoluble in water and also has a strong tendency to adsorb to negatively charged surfaces such as suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many cationic substances in general but long chain alkyl diamines in particular rank among the most difficult substances to test in environmental toxicology. Standard guideline studies are inappropriate to test substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavaibility and exposure assessment for cationic surface-active substances like the diamines as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavaibility that occurs in the environment with cationic surface-active substances.

The aquatic ecotoxicity tests with diamines were therefore performed in river water to allow a PECaquatic,bulk/PNECaquatic,bulkapproach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing 'total aquatic concentrations'. To characterize the risk to the aquatic compartment the PECaquatic,bulkis compared with the PNECaquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2003).

         

In order to class standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method / conditions and test organism used – suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

         

         


For ecotoxicity tests performed using the bulk approach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible. The results of these bulk approach tests are therefore much easier and more realistic, and if compared to PECbulk clearly provide a more appropriate assessment of risks for the environment.

The study was conducted under static conditions with an initial cell density of approximately 5 x 103cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of 2, nominal: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L. Three replicates were tested for the test item concentrations and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Tallowtripropylenetetraminewere analysed at the concentration levels 1.25 and 5.00 mg/L (prepared without algae) and the control at test start and test end via LC-MS/MS analysis. The measured concentrations at test start were in the range of 84 – 93 % of the nominal values. At the end of the test Tallowtripropylenetetraminewas additionally analysed  at  the  concentration  level 1.25 mg/l (prepared  with algae) and  gave  recoveries of

7 - 9 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. Minor adsorption (13 % of nominal) of the test item to glassware was observed. Therefore all effect values are given based on nominal concentrations of the test item.

Tallowtripropylenetetramine (CAS No. 68911-79-5) was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal concentrations): The EC10-values with 95 % confidence intervals for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 1.27 (0.999 – 1.45) and 0.822 (0.758 – 0.894) mg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 2.47 (2.23 – 2.71) and 1.12 (1.04 – 1.18) mg/L, respectively.

After 72 h algae were transferred from the nominal concentration of 1.25 – 10.0 mg/L and control to fresh untreated medium and allowed to grow for further 5 -  7 d under test conditions. The test item effect was observed to be reversible at this concentration. Therefore, there is potential for recovery following exposure up to 10.0 mg/L (highest test concentration)

Description of key information

72 h Algae ErC10 = 1.27 mg/L; 72 h algae ErC50 = 2.47 mg/L

Note that the key algae study is a higher tier study performed in river water with relatively low (realistic worst-case) DOC and suspended matter concentrations. This means that for the risk assessment the bulk approach should be applied i.e. both the effect and exposure assessment are based on the total concentration (dissolved and sorbed).

Key value for chemical safety assessment

EC50 for freshwater algae:
2.47 mg/L
EC10 or NOEC for freshwater algae:
1.27 mg/L

Additional information