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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed acording to OECD guideline n° 201

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-chloropropene
EC Number:
209-187-5
EC Name:
2-chloropropene
Cas Number:
557-98-2
Molecular formula:
C3H5Cl
IUPAC Name:
2-chloroprop-1-ene
Constituent 2
Chemical structure
Reference substance name:
2-chloropropane
EC Number:
200-858-8
EC Name:
2-chloropropane
Cas Number:
75-29-6
Molecular formula:
C3H7Cl
IUPAC Name:
2-chloropropane
Constituent 3
Reference substance name:
1-chloropropene
EC Number:
209-675-8
EC Name:
1-chloropropene
Cas Number:
590-21-6
IUPAC Name:
1-chloroprop-1-ene
Details on test material:
Identification Reaction mass containing mainly 2-chloropropene
Molecular formula C3H5Cl
Molecular weight 76.53
CAS Number 557-98-2
Description Clear light yellow to brown liquid (determined at NOTOX)
Batch RBA100301A
Purity Minimum 60.0%
Test substance storage In refrigerator (2-8°C) in the dark
Stability under storage conditions Stable
Expiry date 08 March 2011 (allocated by NOTOX, 1 year after receipt of the test substance)

Study specific test substance information:
General information Extremely flammable
Test substance handling If necessary, handling after sampling will be performed on dry ice in order to keep the test substance liquefied
Density 0.9
Stability in water: Not indicated
Solubility in water: Yes

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Sampling for analysis of test concentrations:
Duplicate samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0h, t=24h and t=48h
Volume 10 ml
Storage Samples were stored in a freezer until analysis.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period. Additionally, duplicate reserve samples of 10 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of Reaction mass containing mainly 2-chloropropene tested was a clear light yellow to brown liquid with a minimum purity of 60% and the substance was completely soluble in test medium at the concentrations tested.

Because Reaction mass containing mainly 2-chloropropene was volatile, all handlings were performed using air-tight closed test systems.

Preparation of test solutions for the 2nd main test started with a concentration of 1000 mg/l. To this end, 2.22 ml of the test substance was added to 2000 ml medium and the vessel was directly air-tight closed. A 15 minute period of magnetic stirring was applied to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

After preparation, volumes of 75 ml were added to each replicate of the respective test concentration. Subsequently, 1.5 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test System:
Species Pseudokirchneriella subcapitata, strain: NIVA CHL 1

Source In-house laboratory culture.

Reason for selection This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
21-24°C
pH:
between 7.3 and 10.2
Nominal and measured concentrations:
At the start of the test, the actual test concentrations (an average of duplicates) were 0.34, 1.4, 1.9, 3.8 and 8.9 mg/l at the expected nominal concentrations of 4.6, 10, 22, 4.6 and 100 mg/l.
At the end of the 48-hour period the measured concentrations had decreased to 1.0 – 4.8% of the initial concentrations. A similar trend was observed in samples taken from the solution without algae. Based on these results, the Time Weight Average concentrations were calculated to correspond to 0.07, 0.18, 0.46, 095 and 2.1 mg/l.
Details on test conditions:
Fresh water algae culture:
Stock culture 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.

Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/l
K2HPO4.3H2O 52 mg/l
MgSO4.7H2O 75 mg/l
Na2CO3.10H2O 54 mg/l
C6H8O7.H2O 6 mg/l
NH4NO3 330 mg/l
CaCl2.2H2O 35 mg/l
C6H5FeO7.xH2O 6 mg/l
H3BO3 2.9 mg/l
MnCl2.4H2O 1.81 mg/l
ZnCl2 0.11 mg/l
CuSO4.5H2O 0.08 mg/l
(NH4)6Mo7O24.4H2O 0.018 mg/l

Pre-culture 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium M2; according to the OECD 201 Guideline with slightly adjusted NaHCO3 and pH, formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation and with the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 100 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
pH 7.0 ± 0.2

Combined limit / range-finding test:
The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a concentration of 100 mg/l. Test procedure and conditions were similar to those applied in the main tests with the following exceptions:
• Three replicates per concentration were exposed to 0.1, 1.0 and 10 mg/l.
• pH was only measured in the control and the highest test concentration.

Main test 1:
Based on the results of the combined limit/range-finding test a main test 1 was performed. Test procedure and conditions were similar to those applied in the main test 2 with an exception for the concentration range which was: 4.6, 10, 22, 46 and 100 mg/l.

Main test 2:
Test concentrations
Reaction mass
containing mainly 2-chloropropene 10, 32, 100, 320 and 1000 mg/l

Controls Test medium without test substance or other additives

Replicates 6 replicates of the control.
3 replicates of each test concentration.
1 or 2 replicates of each test concentration without algae.
1 replicate of each test concentration for sampling purposes.

Test procedures and conditions
Test duration 48 hours

Test type Static

Test vessels 100 ml, all-glass, containing 75 ml of test solution. Test vessels were closed air-tight with a screw cap and a septum.

Medium M2

Cell density An initial cell density of 1 x 104 cells/ml.

Illumination Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 85 to 97 E.m-2.s-1.

Incubation Vessels were distributed at random in the incubator. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements:
pH At the beginning and at the end of the test.
The pH of the solutions should preferably not deviate by more than 1.5 units during the test.

Temperature of medium Continuously in a temperature control vessel.

Appearance of the cells At the end of the final test microscopic observations were performed to verify a normal and healthy appearance of the inoculum culture and to observe for any abnormal appearance of the algae.

Recording of cell densities:
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with a cuvette (pathlength =10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

Electronic data capture:
Observations/measurements in the study were recorded electronically using the following programme(s):
- Cary 50 single beam spectrophotometer including Cary UVVIS Pharma Upgrade Version 3.1 software (Varian, Mulgrave, Australia): Algal cell density.
- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature.
Reference substance (positive control):
no
Remarks:
Historical controls of Notox with potassium dichromate are included in the report

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
12 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
growth rate
Remarks on result:
other: 95% CL:9.6-15 mg/l
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
4.2 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
other: yield inhibition
Remarks on result:
other: 95% CL:2.2-8 mg/l
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
growth rate
Dose descriptor:
NOEC
Effect conc.:
0.43 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
other: yield inhibition
Details on results:
Combined limit/range-finding test:
Mean cell densities, reduction of growth rate and inhibition of yield
The mean cell densities measured during the combined limit/range-finding test are presented in Table 1. Table 2 presents the percentages growth rate reduction and yield inhibition per concentration. Based on these results samples taken from nominal 10 mg/l were analysed. The initial concentration was 1.9 mg/l. This concentration decreased to 0.21 mg/l at the end of the test (11% of initial). Note, that at the end of the test it was observed that several replicates inadvertently were not air-tight closed. The concentration of the test substance in one of the non air-tight vessels at the end of the test was 0.008 mg/l (0.45% of initial in the 10 mg/l treatment).
The expected EC50 for both growth rate reduction and yield inhibition was beyond the range tested.

All test conditions were maintained within the limits prescribed by the protocol.

Main test 1:
Measured test substance concentrations:

At the start of the test, the actual test concentrations (an average of duplicates) were 0.34, 1.4, 1.9, 3.8 and 8.9 mg/l at the expected nominal concentrations of 4.6, 10, 22, 4.6 and 100 mg/l.
At the end of the 48-hour period the measured concentrations had decreased to 1.0 – 4.8% of the initial concentrations. A similar trend was observed in samples taken from the solution without algae. Based on these results, the Time Weight Average concentrations were calculated to correspond to 0.07, 0.18, 0.46, 095 and 2.1 mg/l.

Mean cell densities
Table 3 shows mean cell densities measured at 24-hour intervals at the different concentrations of Reaction mass containing mainly 2-chloropropene. Table 4 shows the calculation of the percentages of growth rate reduction (total test period) and the percentages of yield inhibition.

Growth rates and yield were in the range of the controls at TWA concentrations of 0.07 mg/l to 0.95 mg/l during the 48-hour test period, whereas the growth rate and yield of algae exposed to the highest concentration was increasingly reduced.

Based on these results it was impossible to calculate the ECx values. Therefore, a main test 2 was performed with a broader concentration range.

All test conditions were maintained within the limits prescribed by the protocol.

Main test 2:
Measured test substance concentrations

At the start of the test, the actual test concentrations (an average of duplicates) were 1.5, 3.8, 14, 59 and 128 mg/l at the expected nominal concentrations of 10, 32, 100, 320 and 1000 mg/l.
At the end of the 48-hour period the measured concentrations had decreased to 1.2 – 9.2% of the initial concentrations. A similar trend was observed in samples taken from the solution without algae. Based on these results, the Time Weight Average concentrations were calculated (see Table 5).

Mean cell densities:
Table 6 shows mean cell densities measured at 24-hour intervals at the different concentrations of Reaction mass containing mainly 2-chloropropene.

Reduction of growth rate and inhibition of yield:
Table 7 shows the calculation of the percentages of growth rate reduction (total test period) and the percentages of yield inhibition. Table 8 shows the calculation of the percentages of growth rate reduction at different time intervals.

Growth rates were in the range of the controls at the TWA concentrations from 0.43 to 2.7 mg/l during the 48-hour test period, whereas the growth rate of algae exposed to 9.7 and 27 mg/l were increasingly reduced. Statistically significant reduction of growth rate was found at TWA concentrations of 2.7 mg/l and higher (Bonferroni t-test, α = 0.05).

Inhibition of yield increased with increasing concentration of Reaction mass containing mainly 2-chloropropene from a TWA concentration of 1.1 mg/l upwards resulting in 97% inhibition at the highest concentration. Statistically significant inhibition of yield was found at test concentrations of 1.1 mg/l and higher (Bonferroni t-test, α = 0.05).

Microscopic observations in the two highest treatments at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Determination of effect concentrations:
Table 9 shows the effect parameters based on TWA concentrations.

Experimental conditions:
Table 10 shows the pH recorded at the beginning and the end of the test. The pH at the beginning of the test was within the limits prescribed by the protocol, whereas at the end of the test exceeded the upper limit. The temperature of the test medium was 22.5°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 21.5 and 22.7°C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

Any other information on results incl. tables

Table 1           Mean cell densities (x104cells/ml) during the
combined limit/range-finding test

Nominal conc.

test substance1

(mg/l)

Exposure time (hours)

 

 

 

0

24

48

control

1.0

4.2

19.4

0.10

1.0

4.7

20.4

1.0

1.0

4.7

17.7

10

1.0

4.6

17.2

100

1.0

4.4

12.3

1.Reaction mass containing mainly 2-chloropropene

 

Table 2           Percentage reduction of growth rate and inhibition of yield during the combined limit/range-finding test

Nominal conc.

test substance1

(mg/l)

Mean growth rate

Yield (0-48 h)

 

 

 

 

µ

(0-48 h)

Reduction

(%)

x104cells/ml

Inhibition (%)

control

0.06162

 

18.43

 

0.10

0.06279

-1.9

19.38

-5.2

1.0

0.05981

2.9

16.69

9.4

10

0.05888

4.5

16.24

11.9

100

0.05222

15.3

11.32

38.6

1.Reaction mass containing mainly 2-chloropropene

 

Table 3           Mean cell densities (x 104cells/ml) during the main test 1

TWA1conc.

Exposure time (hours)

test substance2

 

 

 

(mg/l)

0

24

48

control

1.0

7.0

23.1

0.07

1.0

6.0

21.1

0.18

1.0

4.3

21.6

0.46

1.0

6.2

20.8

0.95

1.0

5.9

20.9

2.1

1.0

5.3

16.2

1.Time Weight Average concentration

2. Reaction mass containing mainly 2-chloropropene

 

Table 4           Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the main test 1

TWA1conc.

test substance2

(mg/l)

Mean growth rate

Yield (0-48 h)

 

 

 

 

µ

(0-48 h)

Reduction

(%)

x104cells/ml

Inhibition (%)

control

0.06538

 

22.09

 

0.07

0.06348

2.9

20.06

9.2

0.18

0.06399

2.1

20.60

6.7

0.46

0.06323

3.3

19.80

10.4

0.95

0.06329

3.2

19.90

9.9

2.1

0.05804

11.2

15.22

31.1

1.Time Weight Average concentration

2. Reaction mass containing mainly 2-chloropropene

 

Table 5           Measured concentrations versus nominal concentrations
in the main test 2

Nominal conc.

test substance1

(mg/l)

Measured concentrations (mg/l) at different time intervals

 

TWA2conc.

(mg/l)

Group mean

TWA2conc.

(mg/l)

t=0h

t=24h

t=48h

10

1.77

0.314

0.0792

0.45

0.43

10

1.32

0.331

0.0909

0.42

32

3.35

0.761

0.236

1.01

1.1

32

4.17

0.864

0.451

1.26

100

12.9

1.11

0.435

2.24

2.7

100

15.7

1.84

0.536

3.18

320

62.3

5.22

0.763

10.01

9.7

320

54.9

5.26

0.673

9.44

1000

116

18.4

3.32

27.01

29

1000

140

20.6

3.4

31.04

1.Reaction mass containing mainly 2-chloropropene

2. Time Weight Average concentration

Table 6           Mean cell densities (x 104cells/ml) during the main test 2

TWA1conc.

Exposure time (hours)

test substance2

 

 

 

(mg/l)

0

24

48

Control

1.0

12.5

57.3

0.43

1.0

12.4

54.8

1.1

1.0

11.3

52.3

2.7

1.0

9.6

44.1

9.7

1.0

4.6

9.7

29

1.0

2.1

2.7

1.Time Weight Average concentration

2. Reaction mass containing mainly 2-chloropropene

Table 7           Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the main test 2

TWA1conc.

test substance2

(mg/l)

Mean growth rate

Yield (0-48 h)

 

 

 

 

µ

(0-48 h)

Reduction

(%)

x104cells/ml

Inhibition (%)

Control

0.08430

 

56.26

 

0.43

0.08342

1.0

53.83

4.3

1.1

0.08242

2.2

51.28

8.8

2.7

0.07888

6.4

43.11

23.4

9.7

0.04738

43.8

8.74

84.5

29

0.02073

75.4

1.74

96.9

1.Time Weight Average concentration

2. Reaction mass containing mainly 2-chloropropene

 

Table 8           Percentage reduction of growth rate at different time intervals during the main test 2

TWA1conc.

test substance2

(mg/l)

Mean growth rate

 

 

 

 

µ

(0-24 h)

Reduction

(%)

µ

(24-48 h)

Reduction

(%)

Control

0.10503

 

0.06357

 

0.43

0.10494

0.1

0.06190

2.6

1.1

0.10082

4.0

0.06402

-0.7

2.7

0.09432

10.2

0.06345

0.2

9.7

0.06343

39.6

0.03133

50.7

29

0.03024

71.2

0.01123

82.3

1.Time Weight Average concentration

2. Reaction mass containing mainly 2-chloropropene

 

Table 9           Effect parameters

Parameter

 

TWA1concentration

Reaction mass containing

mainly 2-chloropropene

(mg/l)

95% confidence interval

(mg/l)

NOERC

1.1

 

48h-ERC10

3.1

2.4 – 3.9

48h-ERC50

12

9.6 – 15

NOEYC

0.43

 

48h-EYC10

1.4

0.70 – 2.7

48h-EYC50

4.2

2.2 – 8.0

1. Time Weight Average concentration

 

  Table 10         pH levels recorded during the main test 2

TWA1conc.

Exposure time (hours)

test substance2

 

 

(mg/l)

0

48

Control

7.4

10.2

0.43

7.4

10.1

1.1

7.3

9.9

2.7

7.3

9.8

9.7

7.3

8.2

29

7.3

8.0

1.Time Weight Average concentration

2. Reaction mass containing mainly 2-chloropropene

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, Reaction mass containing mainly 2-chloropropene reduced growth rate of this fresh water algae species significantly at a TWA concentration of 2.7 mg/l.

The EC50 for growth rate reduction (ERC50: 0-48h) based on TWA concentrations was 12 mg/l with a 95 % confidence interval ranging from 9.6 to 15 mg/l.

The EC50 for yield inhibition (EYC50: 0-48h) based on TWA concentrations was 4.2 mg/l with a 95% confidence interval ranging from 2.2 to 8.0mg/l.

The NOEC for growth rate reduction equalled a TWA concentration of 1.1 mg/l.

The NOEC for yield inhibition equalled a TWA concentration of 0.43 mg/l.
Executive summary:

The study procedures described in this report were based on the OECD guideline No. 201, 2006. In addition, the procedures were designed to meet the test methods of theCommissionRegulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No 761/2009and the ISO International Standard 8692, 2004.

 

The batch of Reaction mass containing mainly 2-chloropropene tested was a clear light yellow to brown liquid with a minimum purity of 60% and the substance was completely soluble in test medium at the concentrations tested.

 

Because Reaction mass containing mainly 2-chloropropene was volatile, all handlings were performed using air-tight closed test systems.

 

Preparation started with a concentration of 1000 mg/l. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

 

Based on the results of a 1stmain test a main test 2 was performed. In the latter test six replicates of exponentially growing algal culture were exposed to a control and three replicates per group were exposed to nominal concentrations of 10, 32, 100, 320 and 1000 mg Reaction mass containing mainly 2-chloropropene per litre. The initial cell density was 104cell/l. Because the test substance was volatile the total exposure time was 48 hours. Samples for analytical confirmation of the actual test concentrations were taken at the start, 24 and 48 hours after test initiation.

 

At the start of the test, the actual test concentrations (an average of duplicates) were 1.5, 3.8, 14, 59 and 128 mg/l at the expected nominal concentrations of 10, 32, 100, 320 and 1000 mg/l.

At the end of the 48-hour period the measured concentrations had decreased to 1.2 – 9.2% of the initial concentrations. A similar trend was observed in samples taken from the solution without algae.Based on these results, the Time Weight Average (TWA) concentrations were calculated to correspond to 0.43, 1.1, 2.7, 9.7 and 29 mg/l.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

Reaction mass containing mainly 2-chloropropene reduced growth rate of the fresh water algae species Pseudokirchneriella subcapitata significantly at a TWA concentration of 2.7 mg/l.

 

The EC50for growth rate reduction (ERC50: 0-48h) based on TWA concentrations was 12 mg/l with a 95 % confidence interval ranging from 9.6 to 15 mg/l.

 

The EC50for yield inhibition (EYC50: 0-48h) based on TWA concentrations was 4.2 mg/l with a 95% confidence interval ranging from 2.2 to 8.0mg/l.

 

The NOEC for growth rate reduction equalled the TWA concentration of 1.1 mg/l.

 

The NOEC for yield inhibition equalled a TWA concentration of 0.43 mg/l.