Pent-1-ene

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:

Experiment 1: 24-27 August 2010. Experiment 2: 07-10 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study under GLP with full documentation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The pH values of the controls and the temperature in the main study and in the reference study did not comply with OECD 201 Guideline. Because the controls showed a normal exponential growth, this can be stated as un-critical.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

See deviation to OECD 201 Guideline. Furthermore, the composition of stock solution II is taken from OECD Guideline 201. As in EU-Method C.3, a different composition (80 mg FeCl3*6H2O/L) is stated, this is stated as a deviation from the EU Method.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

A sample quantity of 1 ml test solution was taken with a syringe and transferred into a GC vial. The vials were immediately closed. After 15 minutes at 20 °C, a sample volume of 50 µl was taken from the headspace and measured via GC/FID.

Vehicle:

no

Details on test solutions:

Pre-culture-medium and Nutrient-medium were prepared according to OECD 201 test guideline

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: CHODAT
- Method of cultivation: Four days before the start of each test, an aliquot of the stock culture containing a few cells was brought into pre-culture medium and incubated for 96 hours. The resulting cul-ture is growing exponentially.
Before usage, the culture was checked on the absence of cell aggregates. After the ad-justment to a cell concentration of about 6*10e+4/mL through photometric measurement and addition of algal medium, the culture was usable for the test. The adjusted pre-culture was mixed with the same amount of 10-fold-nutrient solution. This mixture was the test culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

not reported

Test temperature:

24 – 25 °C experiment 1, 23 – 24 °C experiment 2

pH:

7.0 +/- 0.2
Because the test was performed in a closed system due to the volatility of the test item, the test medium was buffered with NaHCO3 (50 g/L).

Dissolved oxygen:

not reported

Salinity:

not reported

Nominal and measured concentrations:

TEST ITEM
Treatments tested Exp. 1: 0 (control), 10, 22, 46, 100 mg/L
Treatments tested Exp. 2: 0 (control), 13, 18 mg/L
Number of replicates: six replicates for the control, three replicates for each treatment

REFERENCE SUBSTANCE
Treatments tested: 0.04, 0.063, 0.1, 0.16, 0.25, 0.4, 0.63, 1.0, 1.6 mg/L
Number of replicates: six replicates for the control, three replicates for each treatment

Details on test conditions:

TEST SYSTEM
- Test vessel: closed glass flasks closed with Teflon seals and lids, total volume 310 mL
- Type: closed
- Material, size, headspace, fill volume: Total volume 310 mL, fill volume 310 mL
- Aeration: no
- Initial cells density: The algal concentration was adjusted to about 6*10e+4/mL through photometric measurement and addition of algal medium

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: pH adjusted to 7.0 +/- 0.2 with HCl
- Photoperiod: continuous illumination
- Light intensity and quality: 4440 – 8880 Lux

PERFORMANCE OF THE MAIN STUDY (exp. 1 and 2)
The vessels were immediately closed after addition of the test medium, test item and algae, and incubated closed for 72 hours, shaken on an orbital shaker in the lighting incu-bator. To avoid sedimentation of the algal cells, a marble was put into the test vessels. Before start of incubation and every 24 hours, the values of the absorption were recorded. For photometric measurement the seal was transfixed and a volume of 1 ml test solution was taken with a syringe and transferred into a glass cuvette. The hole for sampling was paste up. After photometric measurement the aliquot of the test solution was discarded. After the test, the pH values of the treatments and the control were measured again.
At the end of the test, treatments and control were inspected microscopically in order to verify the appearance of the algae.
The content of the test item in the test vessels was measured at the start and at the end of the test.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (CAS No. 7778-50-9)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

<= 15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

79 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72h EC50 (growth rate): 0.75 mg/L
- 72h EC50 (biomass): 0.26 mg/L
- 72h EC50 (yield): 0.34 mg/L

STABILITY OF THE TEST ITEM IN TEST MEDIUM

The stability in test medium was determined by spiking 259 ml test medium with 0.5 µl (conc. 1.2 mg/L) and 16 µL (conc. 98 mg/L) Pentene-1. After 0 and 3 days storage at room temperature, respectively, 1 ml test solution (two replicates) was collected. A sample volume of 50 µL was taken from the headspace and measured threefold via GC/FID. The solutions in the vials were temperate for 15 minutes at 20°C before measurement. The measured areas at the end of the storage period were compared with the mean areas from the beginning. For each concentration three replicates were analysed.

The results are presented in the following table:

Table 1     Stability in Test Medium at concentration 1.2 mg/L

Time	Area1_1	Area1_2	Area2_1	Area2_2	Area3_1	Area3_2	Area mean
0 days	4.10	4,80	5,79	6,48	6,60	5,86	5,61
3 days	3.76	4,36	3,67	3,91	4,62	5,22	4,26
Recovery after 3 days							75.9 %

 

Table 2     Stability in Test Medium at concentration 98 mg/L

Time	Area1_1	Area1_2	Area2_1	Area2_2	Area3_1	Area3_2	Area mean
0 days	566.12	586,36	622,11	494,46	634,49	666,79	595,06
3 days	362.84	316,40	669,35	474,41	496,71	500,88	470,10
Recovery after 3 days							79.0 %

 

Due to the high volatility of the test item, no better stability could be determined.

RESULTS OF THE MEAIN STUDY WITH THE TEST ITEM

- MAIN STUDY, EXPERIMENT 1

The cell numbers were determined by photometric measurement of optical density. Cell numbers of individual replicates are given in the annex, also. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Table 3     Cell Number/mL Main Study Exp. 1

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
0	Mean	7354	46575	215717	807714
0	SD	0	3798	24902	78988
10	Mean	7354	39221	223071	818745
10	SD	0	4246	23640	21229
22	Mean	7354	44124	164239	671665
22	SD	0	7354	11233	55196
46	Mean	7354	19611	85797	286806
46	SD	0	11233	4246	48223
100	Mean	7354	12257	17159	24513
100	SD	0	4246	4246	4246

SD = Standard Deviation

The pH values in the control ranged from 7.2 to 10.1. The light intensity was in a range of 5200 – 5300 lux. Temperature range was 24 – 25^oC.

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

Table 4     Microscopical Observation Main Study

Nom. Conc. in mg/L	Observation
0	Normal growth
10	Normal growth
22	Normal growth
46	Smaller cells
100	No cells

Because of insufficient homogenisation at the beginning of the test, no test item could be detected in treatment 10 mg/L, whereas the measured concentration in treatment 100 mg/L was much too high. After 72 hours, the measured concentrations were in the expected range. The measured concentration in treatment 10 mg/L was still much higher than expected. As no inhibition in that treatment had been observed, this treatment wasn’t necessary for evaluation of the results.

Therefore, the measured concentrations at the end of the study were used for the determination of the results.

The details are given in the following table:

Table 5     Measured Concentrations in mg/L and corresponding Recovery Rates in % Exp. 1

Nom. Conc.	Measured Conc 0 h	% Nom Conc.	Measured Conc 72 h	% Nom Conc.	% Recovery	geom. Mean.
mg/L	mg/L	%	mg/L	%	%	mg/L
0	n. d.	--	n. d.	--	--	--
10	n. d.	--	18.7	187 %	--	--
22	16.9	77 %	26.9	122 %	159 %	21.3
46	37.8	82 %	38.3	83 %	101 %	38.1
100	282.4	282 %	125.5	125 %	44 %	188.2

n. d. = not detectable

 

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:

Table 6     Growth Rate µ, Area under the Curve AUC, Yield Main Study Exp. 1

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	1.56	647765	800360
		SD	0.03	58943	78988
10	19	Mean	1.57	653280	811391
		SD	0.01	33364	21229
22	27	Mean	1.50	525811	664311
		SD	0.03	31416	55196
46	38	Mean	1.22	230425	279452
		SD	0.06	24483	48223
100	125	Mean	0.40	23288	17159
		SD	0.06	7654	4246

SD = Standard Deviation

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.

Table 7      Inhibition Values Main Study Exp. 1

Nom. Concentration in mg/L	Meas. Concentration in mg/L	% Inhibition
		Growth Rate µ	Area under the Curve AUC	Yield
0	0	0!	0!	0!
10	19	-0.37	-0.85	-1.38
22	27	3.89	18.83	17.00
46	38	22.18	64.43	65.08
100	125	74.56	96.40	97.86

 

- MAIN STUDY (EXPERIMENT 2)

The cell numbers were determined by photometric measurement of optical density. Cell numbers of individual replicates are given in the annex, also. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Table 8     Cell Number/mL Main Study Exp. 2

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
0	Mean	7354	69863	291709	960923
0	SD	0	10137	34073	174937
13	Mean	7354	58832	286806	877577
13	SD	0	14708	65364	292069
18	Mean	7354	51478	245133	769719
18	SD	0	0	25826	128713

SD = Standard Deviation

The pH values in the control ranged from 7.0 to 9.8. The light intensity was in a range of 5200 – 5300 lux. Temperature range was 23 – 24^oC.

The appearance of the alga at the end of the test were "Normal" for the controls and different treatments

Because of insufficient homogenisation the measured concentration at the beginning was much too high. After 72 hours the measured concentrations were in the expected range. The measured concentration in treatment 13 mg/L was marginally higher than measured concentration in treatment 18 mg/L.

The measured concentration at the end of the study was used for the determination of the results.

The details are given in the following table:

Table 9    Measured Concentrations in mg/L and corresponding Recovery Rates in % Exp. 2

Nom. Conc.	Measured Conc. 0 h	% Nom Conc.	Measured Conc. 72 h	% Nom Conc.	% Recovery	geom. Mean.
mg/L	mg/L	%	mg/L	%	%	mg/L
0	n. d.	--	n. d.	--	--	--
13	32.1	247%	17.5	135%	55%	23.7
18	34.8	193%	15.0	83%	43%	22.8

n. d. = not detectable

 

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:

Table 10     Growth Rate µ, Area under the Curve AUC, Yield Main Study Exp. 2

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	1.62	823648	953569
		SD	0.05	118191	174937
13	18	Mean	1.58	766042	870223
		SD	0.13	225149	292069
18	15	Mean	1.55	663086	762365
		SD	0.06	81587	128713

SD = Standard Deviation

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.

Table 11      Inhibition Values Main Study Exp. 2

Nom. Concentration in mg/L	Meas. Concentration in mg/L	% Inhibition
		Growth Rate µ	Area under the Curve AUC	Yield
0	0	0!	0!	0!
13	18	2.52	6.99	8.74
18	15	4.51	19.49	20.05

RESULTS OF THE REFERENCE STUDY

The cell numbers were determined by photometric measurement of optical density. Cell numbers of individual replicates are given in the annex, also.

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.

Table 12     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	-1.23	-0.07	-4.98
0.063	-0.77	8.84	-2.99
0.1	-0.47	-7.88	-1.66
0.16	1.65	5.96	7.42
0.25	11.57	38.04	40.20
0.4	24.10	63.13	65.67
0.63	44.08	85.47	86.27
1.0	62.90	93.97	94.68
1.6	81.36	98.36	98.23

 

The pH values in the control ranged from 8.4 to 8.9. The light intensity was 6200 – 6500 lux. Temperature was 27 °C.

The appearance of the alga at the end of the test were stated. Normal growth was assessed for concentrations <= 0.4 mg/L. No cells were present for concentrations of 0.63 mg/L and above.

Validity criteria fulfilled:

yes

Conclusions:

Toxicity of 1-Pentene to Desmodesmus subspicatus according to OECD 201 resp. EU C.3 was determined. The experiment is valid according to the validity criteria of these guidelines.
The 72h-EC50 (growth rate) is 79 mg/L. The 72h-NOEC is <= 15 mg/L. According to the regulation (CE) 1272/2008, 72h-EC50 (growth rate) should be used for classification.

Executive summary:

Two valid experiments were performed.

Due to the volatility of the test item, the test was performed in a closed system and the nominal load of test item was directly injected into the test vessels.

The first experiment was performed using four concentrations ranging from 10 to 100 mg/L. The second experiment was performed using the concentrations 13 and 18 mg/L. The treatments were used to incubate the unicellular freshwater green algaDesmodesmus subspicatusfor a period of 72 hours. The cell concentration of each replicate was determined by measuring the absorption 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 start and at the end of the test, the content of the test item in the test solutions was determined using GC. The measured values at the end of the test were in the expected range, but at the beginning, the measured values in three treatments were partly too high because of insufficient homogenisation in the completely filled test vessels: a verification experiment in the daphnia study under the same test conditions showed, that after an equilibration time of one hour, test item concentrations in the range of the nominal concentration were determined. Therefore, the determination of the biological results was based on the measured concentration at the end of the test.

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 itemPentene-1were determined:

 

Endpoint	NOEC	LOEC	EC50
Growth Rate	15 mg/L	27 mg/L	79 mg/L
AUC	15 mg/L	27 mg/L	35 mg/L
Yield	15 mg/L	27 mg/L	34 mg/L

 

[1]AUC1 (Area Under Curve according to EU Method C.3) means the integral of the biomass. Calculation see under 8.2.

[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. Calculation see under 8.3

Description of key information

Toxicity to aquatic algae:

OECD 201, GLP, key study, validity 2:

72h-EC50 (growth rate) = 79 mg/L

72h-NOEC (growth rate) <= 15 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

79 mg/L

EC10 or NOEC for freshwater algae:

15 mg/L

Additional information

Two valid experiments were performed.

Due to the volatility of the test item, the test was performed in a closed system and the nominal load of test item was directly injected into the test vessels.

The first experiment was performed using four concentrations ranging from 10 to 100 mg/L. The second experiment was performed using the concentrations 13 and 18 mg/L. The treatments were used to incubate the unicellular freshwater green algaDesmodesmus subspicatusfor a period of 72 hours. The cell concentration of each replicate was determined by measuring the absorption 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 start and at the end of the test, the content of the test item in the test solutions was determined using GC. The measured values at the end of the test were in the expected range, but at the beginning, the measured values in three treatments were partly too high because of insufficient homogenisation in the completely filled test vessels: a verification experiment in the daphnia study under the same test conditions showed, that after an equilibration time of one hour, test item concentrations in the range of the nominal concentration were determined. Therefore, the determination of the biological results was based on the measured concentration at the end of the test.

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

The 72h-EC50 (growth rate) is 79 mg/L. The 72h-NOEC (growth rate) is <= 15 mg/L. According to the regulation (CE) 1272/2008, 72h-EC50 (growth rate) should be used for classification.