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Ecotoxicological information

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:
19 December 2008 to 12 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to GLP and internationally accepted test guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L.
- Sampling method: Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities measured using a Coulter Z Series Particle Count and Size Analyser. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts of uninoculated dilution media. The presence of any abnormal cells was also noted during screening of each test level by microscopic examination.
- Sample storage conditions before analysis: dark, room temperature
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular green alga
- Strain: CCAP 278/4
- Method of cultivation: The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25 °C. These primary cultures provided an inoculum for use in the test (see protocol deviations) in the log phase of growth, characterised by a cell density of 2.19 x 106 cells/mL. Sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and OECD Procedure 201.

ACCLIMATION
- Acclimation period: 3 days
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
The test temperature in the non-adjusted media ranged from 22.4°C to 23.3°C.
In the pH-adjusted media, the temperature ranged from 22.0°C to 23.8°C.
pH:
In non-adjust media, the pH ranged from 7.62 up to 9.87 and varied among the test concentrations and over the duration of the test.
At the start of the test, the control pH was 7.44 and was 8.32 after 72h.
When the pH of the test concentration started below 8, it increased during the test period. For test substances whose initial pH was greater than 8, the pH decreased during the test period.

For pH-adjusted media, the range of starting pH was 7.4 to 7.58, with pH increasing in all concentrations over the duration of the test up to 9.18.
Nominal and measured concentrations:
At the start of the test, the measured levels of 1,3-bis(aminomethyl)cyclohexane in samples of the test cultures ranged between 71 and 108% of their nominal values in both non-adjusted and pH-adjusted media.
After 72 hours, the measured levels ranged between 34 and 111% of their nominal values (between 33 and 113% of their starting values), with losses evident at nominal concentrations of 1.94 to 9.39 mg/L in both non-adjusted and pH-adjusted media.

The overall mean measured levels of 1,3-bis(aminomethyl)cyclohexane were 1.27, 2.97, 5.63, 13.7, 39.2 and 104 mg/L in non-adjusted media and 1.44, 3.47, 5.59, 14.4, 45.8 and 100 mg/L in pH-adjusted media. These results indicate adjustment of pH had no influence on achieved 1,3-bis(aminomethyl)cyclohexane concentrations.

After 72 hours, analysis of samples of media containing 1,3-bis(aminomethyl)cyclohexane at nominal concentrations of 1.94 mg/L, which had been incubated without algal cells, indicated maintenance of test concentrations when compared to test media incubated in the presence of algal cells (83 and 100% of starting concentrations compared to 67 and 73%).
At nominal concentrations of 100 mg/L, results of analysis showed similar results to test media incubated in the presence of algal cells (102 and 115% of starting concentrations). These results indicate that the presence of algal cells affected the stability of the test substance in aquatic media.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 250mL conical flasks loosely stoppered with foam bungs, containing 100mL of test culture or control.
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): static
- Initial cells density: 0.930 x104/mL and 0.750x104
- Control end cells density: 1121406 and 1286178
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):6

GROWTH MEDIUM
- Standard medium used: yes
As recommended in Official Journal No. L383A Part C.3 and OECD Procedue 201
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
56.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL42.2 & 64.4
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
29.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL, 25.0 & 37.4 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: pH Adjusted media; 95% CL, >100 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
58.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: pH Adjusted; 95% CL 43.2 & 75.8 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
25 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 19.0 & 36.3mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL, 9.24 &31.2
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
42.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: pH-adjusted media; 95% CL, 31.0& 54.4 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
16.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: pH adjusted; 95%CL 6.15 & 32.9 mg/L
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
39.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
39.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
45.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: pH-adjusted media
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
13.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
13.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
14.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: pH-Adjusted media
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- 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
- Effect concentrations exceeding solubility of substance in test medium: no

Non-adjusted media
Area under the growth curve
EbC10 (72 h) : 13.8 mg/L (95% confidence limits, 9.24 & 31.2 mg/L)
EbC50 (72 h) : 29.7 mg/L (95% confidence limits, 25.0 & 37.4 mg/L)
No observed effect concentration (NOEC) : 13.7 mg/L
Lowest observed concentration (LOEC) : 39.2 mg/L
Average specific growth rate
ErC10 (0 - 72 h) : 25.0 mg/L (95% confidence limits, 19.0 & 36.3 mg/L)
ErC50 (0 - 72 h) : 56.7 mg/L (95% confidence limits, 42.2 & 64.4 mg/L)
No observed effect concentration (NOEC) : 13.7 mg/L
Lowest observed concentration (LOEC) : 39.2 mg/L
The mean coefficient of variation (CoV) for daily growth rates in non-adjusted control cultures ranged between 5.00 and 11.0% during the definitive test and the CoV for the average specific growth rates of control culture was 3.14% during the 72-hour exposure period.

pH-adjusted media
Area under the growth curve
EbC10 (72 h) : 16.5 mg/L (95% confidence limits, 6.15 & 32.9 mg/L)
EbC50 (72 h) : 58.4 mg/L (95% confidence limits, 43.2 & 75.8 mg/L)
No observed effect concentration (NOEC) : 14.4 mg/L
Lowest observed concentration (LOEC) : 45.8 mg/L
Average specific growth rate
ErC10 (0 - 72 h) : 42.3 mg/L (95% confidence limits, 31.0 & 54.4 mg/L)
ErC50 (0 - 72 h) : >100 mg/L (95% confidence limits, >100 mg/L)
No observed effect concentration (NOEC) : 14.4 mg/L
Lowest observed concentration (LOEC) : 45.8 mg/L
The mean coefficient of variation (CoV) for daily growth rates in pH-adjusted control cultures ranged between 1.78 and 16.3% during the definitive test and the CoV for the average specific growth rates of control culture was 4.24% during the 72-hour exposure period.

These results suggest that the increase in pH caused by the presence of 1,3-bis(aminomethyl)cyclohexane was partially responsible for the higher level of inhibition observed in the non-adjusted media when compared to the pH-adjusted media.

a) Cell densities - non-adjusted media

Exposure concentrations

(mg 1,3-bis(aminomethyl)cyclohexane/L)

 

Replicate number

Cell densities (cells/mL)

 

Nominal

Measured

 

24 hours

48 hours

72 hours

Control

nd

R1

50200

274267

1170517

R2

48100

248667

1202083

R3

48067

283800

1248583

R4

50600

263233

1258517

R5

35200

218700

961317

R6

36733

169800

887417

Mean

44817

243078

1121406

1.94

1.27

R1

44533

242833

1082417

R2

45167

243733

1068783

R3

48667

271900

1195750

Mean

46122

252822

1115650

4.27

2.97

R1

44267

252900

1108083

R2

49767

270633

1183417

R3

47267

235967

1098950

Mean

47100

253167

1130150

9.39

5.63

R1a

30033

106233

502017

R2

40367

240567

1131683

R3

48333

266467

1082350

Mean

44350

253517

1107017

20.7

13.7

R1

41400

212333

983483

R2

45500

244833

1026983

R3

46133

241867

949983

Mean

44344

233011

986817

45.5

39.2

R1

18733

81000

296083

R2

19733

104867

414517

R3

20000

85333

312450

Mean

19489

90400

341017

100

104

R1

5200

7567

8750

R2

4800

6067

5483

R3

6867

5933

4850

Mean

5622

6522

6361

nd : none detected (less than the limit of detection of 0.5 mg/L).

R1-R6 : replicate number.

a : cell counts appear anomalous therefore vessel disregarded from analysis

Note : the initial cell density was estimated to be 0.930 x 104/mL

b) Cell densities - pH-adjusted media

Exposure concentrations

(mg 1,3-bis(aminomethyl)cyclohexane/L)

 

Replicate number

Cell densities (cells/mL)

 

Nominal

Measured

 

24 hours

48 hours

72 hours

Control

nd

R1

50233

295067

1401517

R2

48867

282633

1344483

R3

50200

287000

1328817

R4

27300

156200

835750

R5

49767

292600

1427883

R6

49867

269400

1378617

Mean

46039

263817

1286178

1.94

1.44

R1

51467

279700

1292517

R2

53933

282967

1414183

R3

53933

290567

1366350

Mean

53111

284411

1357683

4.27

3.47

R1

50900

266100

1189350

R2

52467

274833

1078783

R3

45800

263767

1165217

Mean

49722

268233

1144450

9.39

5.59

R1

47667

263167

1268917

R2

50400

276567

1409117

R3

50467

283733

1454550

Mean

49511

274489

1377528

20.7

14.4

R1

43033

238967

1094583

R2

47533

244100

1139750

R3

42633

232233

954350

Mean

44400

238433

1062894

45.5

45.8

R1

41633

210367

937750

R2

38133

194667

644950

R3

43567

214033

736483

Mean

41111

206356

773061

100

100

R1

25867

104867

268817

R2

21167

92833

243917

R3

25733

106433

263083

Mean

24256

101378

258606

nd : none detected (less than the limit of detection of 0.5 mg/L).

R1-R6: replicate number.

Note : the initial cell density was estimated to be 0.750 x 104/mL

a) Inhibition of growth - non-adjusted media

Parameter

Concentration

(mg/l)

Sample

Mean

% Inhibition

p

Nominal

Measured

size

 

 

 

Area under curve to 72 hours

Control

nd

6

19.8

0.0

-

1.94

1.27

3

20.0

-1.0

>0.999W

4.27

2.97

3

20.2

-2.0

>0.999W

9.39

5.63

2

19.8

-0.3

>0.999W

20.7

13.7

3

17.9

9.5

0.221W

45.5

39.2

3

6.1

69.0

<0.001***W

100

104

3

-0.2

101.2

<0.001***W

Growth rate to 72 hours

Control

nd

6

0.065

0.0

-

1.94

1.27

3

0.065

-0.1

>0.999W

4.27

2.97

3

0.066

-0.3

>0.999W

9.39

5.63

2

0.065

0.1

>0.999W

20.7

13.7

3

0.064

2.5

0.383W

45.5

39.2

3

0.049

25.3

<0.001***W

100

104

3

-0.007

110.3

<0.001***W

Growth rate 0 - 24 hours

Control

nd

6

0.062

0.0

-

1.94

1.27

3

0.064

-2.6

>0.999W

4.27

2.97

3

0.065

-4.0

>0.999W

9.39

5.63

2

0.062

0.2

>0.999W

20.7

13.7

3

0.062

0.1

>0.999W

45.5

39.2

3

0.028

55.2

<0.001***W

100

104

3

-0.024

139.5

<0.001***W

Growth rate 24 - 48 hours

Control

nd

6

0.070

0.0

-

1.94

1.27

3

0.071

-0.8

>0.999W

4.27

2.97

3

0.070

0.3

>0.999W

9.39

5.63

2

0.073

-3.5

>0.999W

20.7

13.7

3

0.069

1.7

0.973W

45.5

39.2

3

0.064

9.4

0.108W

100

104

3

0.006

90.8

<0.001***W

 

 

Growth rate 48 - 72 hours

Control

nd

6

0.064

0.0

-

1.94

1.27

3

0.062

3.3

0.590W

4.27

2.97

3

0.062

2.4

0.590W

9.39

5.63

2

0.061

3.9

0.538W

20.7

13.7

3

0.060

5.8

0.220W

45.5

39.2

3

0.055

13.8

0.004**W

100

104

3

-0.002

103.4

<0.001***W

pvalues are for the comparison with Control using Williams' test (W)

**p< 0.01, ***p< 0.001

nd : none detected (less than the limit of detection of 0.5 mg/L).

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to 1,3-bis(aminomethyl)cyclohexane in non-adjusted media, the EbC50 and ErC50, respectively were 29.7 and 56.7 mg/L. In pH-adjusted media, the EbC50 and ErC50, respectively were 58.4 and >100 mg/L.

The following values were derived from the data.
Parameter (mg/L) Non-adjusted pH-adjusted
Area under the growth curve: EbC50 (72 h) 29.7 58.4
95% confidence limits 25.0 & 37.4 43.2 & 75.8
“No observed effect concentration” (NOEC) 13.7 14.4
Area under the growth curve: ErC50 (0 - 72 h) 56.7 > 100
95% confidence limits 42.2 & 64.4 > 100
“No observed effect concentration” (NOEC) 13.7 14.4

The “no observed effect concentration” (NOEC) for both area under the growth curve and growth rate in non-adjusted media was 13.7 mg/L. For, pH-adjusted media the NOEC for both area under the growth curve and growth rate was 14.4 mg/L.
These results suggest that the increase in pH caused by the presence of 1,3-bis(aminomethyl)cyclohexane was partially responsible for the higher level of inhibition observed in non-adjusted media.
Executive summary:

The effect of 1,3-bis(aminomethyl)cyclohexane on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions. The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. No. L383A, 1992) Part C, Method 3 “Algal Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (adopted 2006). Triplicate algal cultures, with an initial cell density of 1 x 104/mL, were exposed to 1,3- bis(aminomethyl)cyclohexane dissolved in algal nutrient medium at nominal concentrations of 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L. The test media were prepared in OECD medium by serial dilution of an aqueous solution. During conduct of the rangefinding test, it was observed that addition of the test substance resulted in an increase in pH. In the definitive test, this effect was observed at nominal concentrations of 9.39 to 100 mg/L. Therefore, two batches of media were prepared. The first batch was tested without adjustment of pH; for the second batch of media, their pH was adjusted to within the range of 7.4 to 7.8, using 0.1M hydrochloric acid. The cultures were incubated in orbital incubators under continuous illumination at temperatures ranging from 21.7 to 23.2°C for 72 hours.

The measured levels of 1,3-bis(aminomethyl)cyclohexane in samples of test cultures ranged between 71 and 108% of their nominal values in freshly prepared non-adjusted and pH adjusted media. During the test, losses were observed at the lower test concentrations, with measured levels in expired (72 hour old) media decreasing to between 34 and 71% of nominal at 1.94 to 9.39 mg/L. At 20.7 to 100 mg/L, measured levels in expired media ranged between 62 and 111% of nominal. The overall mean measured levels of 1,3-bis(aminomethyl)cyclohexane were 1.27, 2.97, 5.63, 13.7, 39.2 and 104 mg/L in non-adjusted media and 1.44, 3.47, 5.59, 14.4, 45.8 and 100 mg/L in pH-adjusted media. These results indicate adjustment of pH had no influence on achieved 1,3-bis(aminomethyl)cyclohexane concentrations. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate.

The following values were derived from the data.

Parameter (g/L)

Non-adjusted

pH-adjusted

 

 

 

Area under the growth curve: EbC50(72h)

29.7

58.4

95% Confidence limits

25.0 & 37.4

43.2 & 75.8

“No observed effect concentration” (NOEC)

13.7

14.4

 

 

 

Area under the growth curve: ErC50(0-72h)

56.7

>100

95% Confidence limits

42.2 & 64.4

>100

“No observed effect concentration” (NOEC)

13.7

14.4

These results suggest that the increase in pH caused by the presence of 1,3- bis(aminomethyl)cyclohexane was partially responsible for the higher level of inhibition observed in the non-adjusted media when compared to the pH-adjusted media.

Description of key information

Algal growth inhibition, 72 hours; EbC50 = 29.7 mg/L, ErC50 = 56.7 mg/L.  In pH-adjusted media over the same period, EbC50 value was found to be 58.4 mg/L and ErC50 was >100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
56.7 mg/L
EC10 or NOEC for freshwater algae:
13.7 mg/L

Additional information

The effect of 1,3-bis(aminomethyl)cyclohexane on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions. The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. No. L383A, 1992) Part C, Method 3 “Algal Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (adopted 2006). Triplicate algal cultures, with an initial cell density of 1 x 104/mL, were exposed to 1,3- bis(aminomethyl)cyclohexane dissolved in algal nutrient medium at nominal concentrations of 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L. The test media were prepared in OECD medium by serial dilution of an aqueous solution. During conduct of the rangefinding test, it was observed that addition of the test substance resulted in an increase in pH. In the definitive test, this effect was observed at nominal concentrations of 9.39 to 100 mg/L. Therefore, two batches of media were prepared. The first batch was tested without adjustment of pH; for the second batch of media, their pH was adjusted to within the range of 7.4 to 7.8, using 0.1M hydrochloric acid. The cultures were incubated in orbital incubators under continuous illumination at temperatures ranging from 21.7 to 23.2°C for 72 hours. The measured levels of 1,3-bis(aminomethyl)cyclohexane in samples of test cultures ranged between 71 and 108% of their nominal values in freshly prepared non-adjusted and pH adjusted media. During the test, losses were observed at the lower test concentrations, with measured levels in expired (72 hour old) media decreasing to between 34 and 71% of nominal at 1.94 to 9.39 mg/L. At 20.7 to 100 mg/L, measured levels in expired media ranged between 62 and 111% of nominal. The overall mean measured levels of 1,3-bis(aminomethyl)cyclohexane were 1.27, 2.97, 5.63, 13.7, 39.2 and 104 mg/L in non-adjusted media and 1.44, 3.47, 5.59, 14.4, 45.8 and 100 mg/L in pH-adjusted media. These results indicate adjustment of pH had no influence on achieved 1,3-bis(aminomethyl)cyclohexane concentrations. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate.

The following values were derived from the data.

Parameter (g/L)

Non-adjusted

pH-adjusted

 

 

 

Area under the growth curve: EbC50(72h)

29.7

58.4

95% Confidence limits

25.0 & 37.4

43.2 & 75.8

“No observed effect concentration” (NOEC)

13.7

14.4

 

 

 

Area under the growth curve: ErC50(0-72h)

56.7

>100

95% Confidence limits

42.2 & 64.4

>100

“No observed effect concentration” (NOEC)

13.7

14.4

These results suggest that the increase in pH caused by the presence of 1,3- bis(aminomethyl)cyclohexane was partially responsible for the higher level of inhibition observed in the non-adjusted media when compared to the pH-adjusted media.