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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 February 2018 to 3rd July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: 1,3-dichloro-2-propanol
Appearance: Colourless liquid
Batch: ZMG-197685
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature container flushed with
nitrogen
Stable under storage conditions until: 29 December 2018 (expiry date)
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
The batch of 1,3-dichloro-2-propanol tested was a colourless liquid with a purity of 99.2%.
No correction was made for the purity/composition of the test item. Preparation of the stock
solution was performed in the glovebox (nitrogen environment).
A stock solution of 10 g/L was prepared by adding 3.7 mL tests item, corresponding with
5.0 g, to 500 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp.,
Bedford, Mass., USA). Magnetic stirring for 18-22 minutes was applied to accelerate
dissolution and to ensure homogeneity.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test system: Micro-organisms in activated sludge.

Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas',
's-Hertogenbosch, The Netherlands, receiving predominantly
domestic sewage.

Preparation of the sludge: The sludge was coarsely sieved (1 mm) and allowed to settle.
The supernatant was removed and ISO-medium was added. A
small amount of the sludge was weighed and dried overnight at
ca. 105°C to determine the amount of suspended solids (3.0 g/L
of sludge, as used for the test). The pH was 7.2 (combined
limit/range-finding test) or 7.9 (final test) on the day of testing.
The batch of sludge was used one day after collection; therefore
50 mL of synthetic medium (=sewage feed) was added per litre
of activated sludge at the end of the collection day. The sludge
was kept aerated at test temperature until use.

Medium: Adjusted ISO-medium, formulated using RO water (tap water
purified by reverse osmosis; GEON Waterbehandeling, BerkelEnschot,
The Netherlands) with the following composition:

CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Rationale Recognized by international guidelines as the recommended
test system.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
3 hours, during which aeration and stirring took place.
Post exposure observation period:
After the 3-hour contact time the oxygen consumption was
recorded for a period of approximately 10 minutes. During
measurement, the sample was not aerated but continuously
stirred on a magnetic stirrer.
Hardness:
Not recorded
Test temperature:
The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test, and was slightly outside the range prescribed by the study plan (20 ± 2°C
pH:
The pH in all test vessels, before addition of sludge, was between 7.0 and 7.2. After the 3 hour exposure period the pH was between 8.0 and 8.4.
Dissolved oxygen:
the dissolved oxygen concentration at the start was above 60-70% saturation
(60% of air saturation is > 5 mg/L at 20°C)
Salinity:
Not recorded
Conductivity:
Not recorded
Nominal and measured concentrations:
Rangefinding nominal concentrations: 10, 100 and 1000 mg/L
Final test nominal concentrations: 0, 1, 3.2, 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
Contact time: 3 hours, during which aeration and stirring took place.
Vessels: All glass open bottles/vessels.
Milli-RO / Milli-RO water Tap water purified by reverse osmosis (Millipore Corp.,
Bedford, Mass., USA).

Synthetic medium 16 g peptone
(=sewage feed) 11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2..2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4


Dissolved in Milli-RO water, made up to 1 litre
and filtered. The pH was within 7.5 ± 0.5.

Air supply Clean, oil-free air.
Aeration The aeration was adjusted in such a way that the dissolved
oxygen concentration at the start was above 60-70% saturation
(60% of air saturation is > 5 mg/L at 20°C) and to maintain the
sludge flocs in suspension.
Test set up 5 replicates per test group and 6 replicates for the control.

Performance of the test
The synthetic medium (=sewage feed, 16 mL) and an appropriate amount of the test item stock were mixed and made
up to 250 mL with Milli-RO water.
The pH was determined. Thereafter, 250 mL activated sludgewas added. This was the start of the test.
After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During
measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item
concentrations and controls.
The medium temperature was recorded continuously in a temperature control vessels. The temperature control vessels
were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in
each fume cupboard of the climate room. Oxygen recording Determination of oxygen was performed with multiple oxygen
sensors connected to a BlueBox (GO-Systemelektronik GmbH,Germany), a multichannel measuring and controlling system.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
<= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
ca. 18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
ca. 55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
ca. 449 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The combined limit/range-finding showed 15%, 47% and 63% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was between a concentration of 100 and 1000 mg/L.
There was no oxygen uptake from abiotic processes.

Acceptability of the Test
The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (34 mg oxygen per one gram of activated sludge in the final test).
The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test (4%).
The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (6.0 mg/L in the final test).
Since all criteria for acceptability of the test were met, this study was considered to be valid.
All test conditions and acceptability criteria prescribed by the study plan were met.

The effects observed were in agreement with what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC and ECx values.
No statistically significant inhibition of the respiration rate of the sludge was recorded at 10 mg 1,3-dichloro-2-propanol per liter (for more information on statistics see Appendix 4). At higher concentrations the inhibitory effect of 1,3-dichloro-2-propanol on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 18% inhibition at 32 mg/L to 67% at 1000 mg/L.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (6.0 mg/L in the final test).
Reported statistics and error estimates:
Interpretation of results
ECx
For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For the test item, calculation of the ECx value was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.
NOEC determination
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Final Test – Overview of the Results

Treatment

Concentration

(mg/L)

Mean respiration rate

% Inhibition of the respiration rate (mean value)

(mg O2/L h)

(mg O2/g h)¹

Control

0

51.27

34.18

 

T1

1.0

56.00

37.33

-9.23

T2

3.2

51.30

34.20

-0.07

T3

10

49.08

32.72

4.27

T4

32

42.25

28.17*

17.59

T5

100

36.68

24.45*

28.46

T6

320

31.58

21.06*

38.39

T7

1000

17.20

11.47*

66.45

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Combined Limit/Range-Finding Test – Respiration Rate/Inhibition, pH Values

Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)¹

C 1

0

7.4

7.0

43.79

29.19

 

C 2

0

7.4

6.7

41.36

27.57

 

C 3

0

7.4

6.7

44.29

29.53

 

C 4

0

7.4

6.6

44.16

29.44

 

C 5

0

7.4

6.7

41.40

27.60

 

C 6

0

7.4

6.4

46.34

30.89

 

C Mean

 

 

 

43.56

29.04 (RC)

 

SD

 

 

 

1.91

1.27

 

CV (%)

 

 

 

4

4

 

 

 

 

 

 

 

R 1

1.0

7.4

7.0

46.39

30.93

-6.50

R 2

3.2

7.4

7.7

30.94

20.63

28.97

R 3

10

7.4

7.8

10.72

7.15

75.39

R 4

32

7.6

7.8

4.72

3.15

89.16

 

 

 

 

 

 

 

T 1

10

7.4

7.1

37.00

24.67

15.05

T 2

100

7.4

7.4

23.22

15.48

46.69

T 3a

1000

7.4

7.6

15.06

10.04

65.42

T 3b

1000

7.4

7.6

16.39

10.93

62.37

T 3c

1000

7.4

7.6

16.75

11.17

61.54

T3 Mean

 

 

16.07

10.71 (RT)*

63.11 (IT)

 

 

 

 

 

 

 

TA

1000

7.4

7.3

0.20

0.13

99.54

C:          Control

R:          Reference item, 3,5-dichlorophenol       

T:          Test item, 1,3-dichloro-2-propanol        

TA:       Abiotic control of 1,3-dichloro-2-propanol            

SD:       Standard deviation  

CV:       Coefficient of variation                          

RT:         Total respiration with 1,3-dichloro-2-propanol

RC:         Total respiration in the control

IT:           % inhibition of total respiration relative to RC

¹             The amount of suspended solids in the final test mixture was 1.5 g/L.

*            Statistically significantly different compared to control       

Table6          
Final Test –Respiration Rate/Inhibition, pH Values


Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)¹

C 1

0

7.0

8.1

50.55

33.70

 

C 2

0

7.0

8.2

47.62

31.75

 

C 3

0

7.0

8.1

52.01

34.67

 

C 4

0

7.0

8.1

51.97

34.65

 

C 5

0

7.0

8.1

53.50

35.67

 

C 6

0

7.0

8.1

51.94

34.63

 

C Mean

 

 

 

51.27

34.18 (RC)

 

SD

 

 

 

2.01

1.34

 

CV (%)

 

 

 

4

4

 

 

 

 

 

 

 

 

R 1

1.0

7.0

8.1

49.83

33.22

2.80

R 2

3.2

7.0

8.2

34.17

22.78

33.35

R 3

10

7.1

8.3

16.19

10.79

68.42

R 4

32

7.2

8.4

8.88

5.92

82.68

 

 

 

 

 

 

 

T 1a

1.0

7.0

8.0

62.43

41.62

-21.78

T 1b

1.0

7.0

8.0

57.76

38.51

-12.67

T 1c

1.0

7.0

8.0

55.22

36.81

-7.71

T 1d

1.0

7.0

8.0

53.11

35.41

-3.60

T 1e

1.0

7.0

8.1

51.47

34.31

-0.40

T 1 Mean

 

 

 

56.00

37.33 (RT)

-9.23 (IT)

 

 

 

 

 

 

 

T 2a

3.2

7.0

8.1

53.09

35.39

-3.56

T 2b

3.2

7.0

8.1

52.50

35.00

-2.41

T 2c

3.2

7.0

8.1

51.07

34.05

0.38

T 2d

3.2

7.0

8.1

49.59

33.06

3.27

T 2e

3.2

7.0

8.1

50.26

33.51

1.96

T 2 Mean

 

 

 

51.30

34.20 (RT)

-0.07 (IT)

C:        Control

SD:    Standard deviation

CV:     Coefficient of variation

R:        Reference item, 3,5-dichlorophenol

T:        Test item, 1,3-dichloro-2-propanol

¹:         The amount of suspended solids in the final test mixture was 1.5 g/L

RC:      Total respiration in the control

RT:      Total respiration with 1,3-dichloro-2-propanol

IT:        % inhibition of total respiration relative to RC


  Final Test – Respiration Rate/Inhibition, pH Values

Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)¹

T 3a

10

7.0

8.1

50.87

33.91

0.77

T 3b

10

7.0

8.1

48.38

32.25

5.63

T 3c

10

7.0

8.0

57.75

38.50

-12.65

T 3d

10

7.0

8.2

41.16

27.44

19.71

T 3e

10

7.0

8.1

47.23

31.49

7.87

T 3 Mean

 

 

 

49.08

32.72 (RT)

4.27 (IT)

 

 

 

 

 

 

 

T 4a

32

7.0

8.2

45.14

30.09

11.95

T 4b

32

7.0

8.2

41.16

27.44

19.71

T 4c

32

7.0

8.1

43.10

28.73

15.93

T 4d

32

7.0

8.2

39.36

26.24

23.22

T 4e

32

7.0

8.1

42.48

28.32

17.14

T 4 Mean

 

 

 

42.25

28.17 (RT)*

17.59 (IT)

 

 

 

 

 

 

 

T 5a

100

7.0

8.3

33.69

22.46

34.28

T 5b

100

7.0

8.2

40.32

26.88

21.35

T 5c

100

7.0

8.1

39.43

26.29

23.09

T 5d

100

7.0

8.2

37.21

24.81

27.42

T 5e

100

7.0

8.2

32.73

21.82

36.16

T 5 Mean

 

 

 

36.68

24.45 (RT)*

28.46 (IT)

 

 

 

 

 

 

 

T6a

320

7.0

8.2

33.95

22.63

33.78

T6b

320

7.0

8.2

31.24

20.83

39.06

T6c

320

7.0

8.2

29.15

19.43

43.14

T6d

320

7.0

8.1

32.33

21.55

36.94

T6e

320

7.0

8.2

31.25

20.83

39.04

T6 Mean

 

 

 

31.58

21.06 (RT)*

38.39 (IT)

 

 

 

 

 

 

 

T7a

1000

7.0

8.3

19.99

13.33

61.01

T7b

1000

7.0

8.4

17.16

11.44

66.53

T7c

1000

7.0

8.3

15.61

10.41

69.55

T7d

1000

7.0

8.4

14.89

9.93

70.95

T7e

1000

7.0

8.3

18.34

12.23

64.23

T7 Mean

 

 

 

17.20

11.47 (RT)*

66.45 (IT)

T:        Test item, 1,3-dichloro-2-propanol

¹:         The amount of suspended solids in the final test mixture was 1.5 g/L

RT:      Total respiration with 1,3-dichloro-2-propanol

IT:        % inhibition of total respiration relative to RC

*:         Statistically significantly different compared to control

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of this present test 1,3-dichloro-2-propanol was not toxic to waste water bacteria (activated sludge) at or below a concentration of 10 mg/L (NOEC).
The EC10 was 18 mg/L (95% confidence interval: 12 - 26 mg/L).
The EC20 was 55 mg/L (95% confidence interval: 41 - 70 mg/L).
The EC50 was 449 mg/L (95% confidence interval: 371 - 557 mg/L).

Executive summary:

The objective of the study was to evaluate 1,3 -dichloro-2-propanol for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50and/or the no - observed effect concentration (NOEC).

The influence of 1,3-dichloro-2-propanol on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based onthe OECD guideline No. 209, 2010.

The batch of 1,3-dichloro-2-propanol tested was a colourless liquid with a purity of 99.2%. No correction was made for the purity/composition of the test item. Preparation of the stock solution was performed in the glovebox (nitrogen environment).

The test started with the preparation of a stock solution of 10 g/L. Magnetic stirring was applied to accelerate dissolution and to ensure homogeneity. Volumes ofthe clear and colourlessstock solution corresponding to the test concentration were then added to the test media. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The final test was performed based on the result of a preceding combined limit/range-finding test. Seven concentrations were tested, ranging from 1.0 to 1000 mg/L and increasing with a factor 3.2. Five replicates per concentration and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 10 mg 1,3-dichloro-2-propanol per liter. At higher concentrations the inhibitory effect of  1,3-dichloro-2-propanol on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 18% inhibition at 32 mg/L to 67% at 1000 mg/L.

The batch of activated sludge was tested for sensitivity with the reference item
3,5-dichlorophenol, and showed normal sensitivity.

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

1,3-dichloro-2-propanol was not toxic to waste water (activated sludge) bacteria at a concentration of 10 mg/L (NOEC).

The effect parameters for the inhibition of the respiration rate are presented below.

Parameter

1,3-dichloro-2-propanol

Concentration (mg/L)

NOEC

10

EC10

18 (12 - 26 mg/L)

EC20

55 (41 - 70 mg/L)

EC50

449 (371 - 557 mg/L)

 (  ) Between brackets the 95% confidence intervals are given.

Description of key information

Under the conditions of this present test 1,3-dichloro-2-propanol was not toxic to waste water bacteria (activated sludge) at or below a concentration of 10 mg/L (NOEC).

The EC10 was 18 mg/L (95% confidence interval: 12 - 26 mg/L).

The EC20 was 55 mg/L (95% confidence interval: 41 - 70 mg/L).

The EC50 was 449 mg/L (95% confidence interval: 371 - 557 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:
449 mg/L
EC10 or NOEC for microorganisms:
10 mg/L

Additional information