2-propanol and 2-butanol production, distn. residues

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September - 17 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully documented study run under GLP and according to international guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Temperature in reference study and main study higher as in guideline. pH values of the controls in main study varied more than stated in the guideline. As the controls showed a normal exponential growth, these deviations can be stated as uncritical.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

Temperature in reference study and main study higher as in guideline. pH values of the controls in main study varied more than stated in the guideline. As the controls showed a normal exponential growth, these deviations can be stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the beginning and at the end of the test, the content of test item was determined by DOC-determination in the test solutions.

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CHODAT
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Age of inoculum (at test initiation): From September 2010
- Method of cultivation: The algae are kept as stock culture on solid agar at 8 °C. From an aliquot of this stock culture the preculture was prepared.

ACCLIMATION
- Acclimation period: Four days before the start of each test, an aliquot of the stock culture containing a few cells was brought into preculture medium and incubated for 72 hours. The resulting culture is growing exponentially.
Before usage, the culture was checked on the absence of cell aggregates. After the adjustment to a cell concentration of about 6*10 E4/mL through photometric measurement and addition of algal medium, the culture was usable for the test. The adjusted preculture was mixed with the same amount of 10-fold-nutrient solution. This mixture was the test culture.
- Culturing media and conditions (same as test or not): same as test (without substance)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 - 27 °C

pH:

7 - 9.5 (in controls 7 - 9)

Nominal and measured concentrations:

Nominal and measured concentrations: See table in "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: closed glass beakers
- Type (delete if not applicable):closed
- Material, size, headspace, fill volume: glass, 130 mL
- Aeration: no (indirectly by shaking)
- Initial cells density: 6*10^4/mL
- Control end cells density:487*10^4/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: all 24h

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no: no information
- Adjustment of pH: To avoid a high pH increase, 700 ml deionised water were mixed with 6 mL sodium hydrogencarbonate solution (50 g/L). Then, pH was adjusted to 7.0 (±0.2) with HCl.
- Photoperiod: 72 hours
- Light intensity and quality: 5800 – 5900 Lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
- Chlorophyll measurement: no

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

dissolved organic carbon

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

dissolved organic carbon

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

dissolved organic carbon

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

dissolved organic carbon

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

dissolved organic carbon

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

dissolved organic carbon

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

58 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

dissolved organic carbon

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

dissolved organic carbon

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

dissolved organic carbon

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

> 63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: dissolved organic carbon

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

> 63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: dissolved organic carbon

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

> 63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: dissolved organic carbon

Basis for effect:

other: yield

Results with reference substance (positive control):

NOEC 72 h 0.4 mg/L
LOEC 72 h 0.63 mg/L
72h ErC50 2.0 mg/L
72h EbC50 0.49 mg/L
72h EyC50 0.34 mg/L

Reported statistics and error estimates:

For the treatments with the nominal test item concentrations 18 and 32 mg/L (measured concentrations 8.6 and 18.7 mg/L), it was tested whether the differences between treatment and control were significant. The values for the area under the curve, yield and the growth rate were used for the assessment of observed effect levels.
In order to select a suitable test for significance, it was checked whether equality of variance was given.
The calculated value F is compared with the F-test table (level of significance 95%). If the calculated value is smaller than the tabular value, equality of variance is given. If equality of variance is given, the t-test is used; else, the WEIR test is used. With the t-Test it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table with grade of freedom: n1 + n2 – 2, level of significance 95%).

Determination of the NOEC
For the endpoints µ, AUC and yield, the differences between the treatment 18 mg/L (measured concentration 8.6 mg/L) and the control can be considered as not significant (level of significance: 97.5%) as the calculated t-values were smaller than the limit of significance. Therefore, the measured concentration 8.6 mg/L is stated as NOEC for these endpoints.

Determination of the LOEC
For the endpoints µ, AUC and yield, the differences between the treatment 32 mg/L (measured concentration 18.7 mg/L) and the control must be considered as significant (level of significance: 97.5%), the measured concentration 18.7 mg/L can be stated as LOEC for these endpoints.

The estimation of the EC50s of the test item was accomplished using the software OriginTM.

Main Study

Cell Numbers

The cell numbers were determined by photometric measurement of optical density. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Cell Number/mL Main Study

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
0	Mean	7354	24513	106633	486590
0	SD	0	6005	12084	48873
10	Mean	7354	22062	115213	563807
10	SD	0	0	22467	88350
18	Mean	7354	24513	151983	720692
18	SD	0	4246	4246	36770
32	Mean	7354	25739	73540	290483
32	SD	0	5200	10400	98801
56	Mean	7354	17159	26965	88248
56	SD	0	4246	15309	44733
100	Mean	7354	9805	12257	75991
100	SD	0	4246	8492	29721

SD = Standard Deviation

Temperature, Light Intensity, pH

The pH values in the control ranged from 7.0 to 9.0. The light intensity was in a range of 5800 – 5900 lux. Temperature range was 24.0 – 27.0 °C. The details are given in the following table:

Temperature, Light Intensity, pH Main Study

		0 h	24 h	48 h	72 h
Temperature [°C]		24	25	24	27
Light intensity [lux]		5800	5800	5900	5900
pH	Conc. in mg/L		-	-
	0	7.0	-	-	9.0
	10	7.0	-	-	9.5
	18	7.0			9.5
	32	7.0			9.1
	56	7.0	-	-	7.3
	100	7.0	-	-	7.1

Microscopical Observations

In the following table, the appearance of the alga at the end of the test is stated:

Microscopical Observation Main Study

Nom. Conc. in mg/L	Observation
0	normal growth
10	normal growth
18	normal growth
32	smaller cells
56	no cells
100	no cells

 

Analytical Determination

The measured and calculated concentrations for treatment and control are given in the following table. The concentration of test item was calculated through measured DOC divided by the content of organic carbon (76%) of the test item.

DOC Values and Recovery

Nominal Conc. mg/L	DOC mg/L	Test item mg/L	% of Nominal	DOC mg/L	Test item mg/L	% of Nominal	% Recovery	Geom. Mean of Test Item mg/L
	0h			72h
0	2.34	-	-	2.33	-	-	-	-
10	6.82	8.97	90%	4.37	5.75	58%	64%	7.18
18	6.82	8.97	50%	6.31	8.30	46%	93%	8.63
32	15.25	20.07	63%	13.24	17.42	54%	87%	18.70
56	32.46	42.71	76%	29.03	38.20	68%	89%	40.39
100	54.05	71.12	71%	42.85	56.38	56%	79%	63.32

Area under the Curve, Growth Rate, Yield

From the cell numbers, the Growth Rate µ, the Area under the Curve AUC, and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Growth Rate µ, Area under the Curve AUC, Yield Main Study

Nom. Concentration in mg/L	Meas. Concentration in mg/L		Growth Rate [day^-1]	AUC [Cell Concentration/mL*day]	Yield [Cell Concentration/mL]
0		Mean	1.40	356056	479236
		SD	0.04	41236	48873
10	7.2	Mean	1.44	400793	556453
		SD	0.05	66492	88350
18	8.6	Mean	1.53	518457	713338
		SD	0.02	22963	36770
32	18.7	Mean	1.22	226136	283129
		SD	0.12	65001	98801
56	40.4	Mean	0.80	69863	80894
		SD	0.16	41763	44733
100	63.3	Mean	0.76	41673	68637
		SD	0.12	27598	29721

SD = Standard Deviation

Reference Study

Cell Numbers

The cell numbers were determined by photometric measurement of optical density. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Cell Number/mL Reference Study

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
0	Mean	7354	60303	238270	760404
0	SD	0	9588	69882	187966
0.04	Mean	7354	63735	230425	823648
0.04	SD	0	8492	4246	19457
0.063	Mean	7354	66186	227974	728046
0.063	SD	0	0	7354	74635
0.1	Mean	7354	58832	166691	539293
0.1	SD	0	7354	48965	176700
0.16	Mean	7354	51478	134823	485364
0.16	SD	0	19457	73662	247099
0.25	Mean	7354	56381	149531	436337
0.25	SD	0	4246	74029	276077
0.4	Mean	7354	71089	174045	509877
0.4	SD	0	4246	4246	29721
0.63	Mean	7354	61283	115213	296611
0.63	SD	0	4246	8492	15309
1.0	Mean	7354	53929	83345	98053
1.0	SD	0	4246	18507	16983
1.6	Mean	7354	58832	56381	83345
1.6	SD	0	0	8492	8492

SD = Standard Deviation

 Area under the Curve, Growth Rate, Yield

From the cell numbers, the Growth Rate µ, the Area under the Curve AUC, and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Growth Rate µ, Area under the Curve AUC, Yield Reference Study

Nominal Concentration in mg/L		Growth Rate [day^-1]	AUC [Cell Concentration/mL*day]	Yield [Cell Concentration/mL]
0	Mean	1.54	660389	753050
	SD	0.09	166860	187966
0.04	Mean	1.57	687599	816294
	SD	0.01	18385	19457
0.063	Mean	1.53	639798	720692
	SD	0.03	42405	74635
0.1	Mean	1.42	476784	531939
	SD	0.11	138039	176700
0.16	Mean	1.36	410598	478010
	SD	0.18	215107	247099
0.25	Mean	1.31	405696	428983
	SD	0.22	215327	276077
0.4	Mean	1.41	481687	502523
	SD	0.02	13258	29721
0.63	Mean	1.23	306417	289257
	SD	0.02	18869	15309
1.0	Mean	0.86	167916	90699
	SD	0.06	25826	16983
1.6	Mean	0.81	138500	75991
	SD	0.04	11233	8492

 

 Inhibition

The following mean inhibition values were calculated for the Growth Rate µ, the Area under the Curve AUC, and the yield. Individual inhibition values are given in the annex.

Inhibition Values Reference Study

Nominal Concentration in mg/L	% Inhibition
	Growth Rate µ	Area under the Curve AUC	Yield
0	0!	0!	0!
0.04	-2.36	-4.12	-8.40
0.063	0.39	3.12	4.30
0.1	7.63	27.80	29.36
0.16	11.16	37.82	36.52
0.25	14.56	38.57	43.03
0.4	8.06	27.06	33.27
0.63	19.81	53.60	61.59
1.0	44.01	74.57	87.96
1.6	47.41	79.03	89.91

Validity criteria fulfilled:

yes

Remarks:

except for the pH in the reference study

Conclusions:

The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:
The NOECs of the growth rate, AUC and yield were 8.6 mg/L and the LOECs 19 mg/L. The EC50 value for the growth rate was 58 mg/L, for the AUC 23 mg/L and for the yield also 23 mg/L.
Based on these data in combination with the fact that the substance is not readily biodegradable, a classification into aquatic chronic category 3 (H412) (corresponding to R52/53) is necessary (EC50 band 10 - 100 mg/L).

Executive summary:

The 72h-EC50 of reaction mass of "2-propanol and 2-butanol production, distn. residues" (main constituent 2,2'-oxybisbutane (DSBE), DIPE, SBA and 2 -methylpropan-2 -ol (TBA)) against Desmodesmus subspicatus was determined according to OECD 201 resp. EU C.3.

One valid main study was performed.

The study was performed using five concentrations ranging from 10 to 100 mg/L. The treatments were used to incubate the unicellular freshwater green alga Desmodesmus subspicatus for a period of 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the cuvettes at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate µ, the area under the growth curve (AUC [1]) and the Yield [2] were determined.

At the beginning and at the end of the test, the content of test item was determined by DOC-determination in the test solutions. The content of the test item was calculated from content of organic carbon of the test item (76 %) The recovery after 72 hours ranged from 64 to 93 % of the start concentration. The correlation between nominal and measured concentration was poor. Therefore, the geometric mean of the measured concentration was used for the determination of the biological results and the determination of the biological results was based on the mean of the measured concentrations.

The EC50s of potassium dichromate were tested in a current reference test. The values lay within the normal range of the laboratory.

The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:

Growth Rate: NOEC 8.6 mg/L, LOEC 19 mg/L, EC50 58 mg/L

AUC: NOEC 8.6mg/L, LOEC 19 mg/L, EC50 23 mg/L

Yield: NOEC 8.6mg/L, LOEC 19 mg/L, EC50 25 mg/L

 

[1] AUC1 (Area Under Curve according to EU Method C.3) means the integral of the biomass.

[2] Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period.

Description of key information

The following results were determined for the test item "2-propanol and 2-butanol production, distn. residues" (species: Desmodesmus subspicatus): 72h-ErC50 (growth rate) = 58 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

58 mg/L

EC10 or NOEC for freshwater algae:

8.6 mg/L

Additional information

The EC50 value and the NOEC for growth inhibition, AUC and yield of "2 -propanol and 2 -butanol, distn residues" was measured according to OECD 201, respectively EU C.3 guideline under GLP. Following results were obtained:

Endpoint	NOEC	LOEC	EC50
Growth Rate	8.6 mg/L	19 mg/L	58 mg/L
AUC	8.6 mg/L	19 mg/L	23 mg/L
Yield	8.6 mg/L	19 mg/L	25 mg/L

The analytical determinations of the test item showed poor correlation between nominal and measured concentrations and acceptable recovery rates. The poor correlation is probably caused by the high volatility of the test item. Therefore, the determination of the results of the test item "2 -propanol and 2 -butanol, distn. residues" was based on the geometric means of the measured concentrations.

Based on these data in combination with the fact that the substance is not readily biodegradable, a classification into aquatic chronic category 3 (H412) according to the CLP regulation (corresponding to R52/53) is necessary (EC50 10 - 100 mg/L).

Based on the lowest EC50 value of three species daphnia, algae and fish (available studies), an aquatic PNEC value was calculated for the risk assessment. As values from algae test based on growth rate, not on biomass or yield, should be employed for classification and risk assessment, the relevant EC50 value is 58 mg/L. Therefore, the value for algae does not represent the lowest value of three species daphnia, algae and fish.