Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to an appropriate guideline and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Batch No.: 1503000133
Purity: 94.6%
Appearance: Crystalline Powder
Receipt Date: 26 March 2016
Expiry Date: 03 March 2017
Storage: Ambient

Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
not required
Details on sampling:
Not applicable

Test solutions

Vehicle:
no
Details on test solutions:
The test was conducted at nominal dipentaerythritol concentrations of 10, 100 and 1000 mg/L. Three nominal reference item (5, 15 and 30 mg 3, 5-DCP/L) treatments and two controls were also included in the test.

Test vessels were prepared by the direct addition of pre-weighed dipentaerythritol aliquots. Glass vials were rinsed after addition with deionised water to ensure all test item was removed from the vials. One replicate flask was prepared for the 10 and 100 mg/L test treatments and three replicate flasks for the 1000 mg/L. Test item weights were adjusted for purity prior to weighing.

A 1 g/L reference item stock solution was prepared by adding 3, 5-DCP (249.90 mg) to a glass volumetric flask (250 mL) containing an amount of deionised water. The solution was sonicated for ca 20 min to aid dissolution. The pH of the stock solution was measured and adjusted to be between pH 7-8 using 1M NaOH prior to being brought up to volume with deionised water. Aliquots were added to the appropriate reference vessels to give the required addition rates. One replicate flask was prepared for each reference treatment.

No test or reference item was added to the control vessels. Two replicate flasks were prepared for the control treatment.

Prior to the test initiation synthetic sewage feed was combined with reference or test item as appropriate. Deionised water was then added to provide 250 mL per flask final volume.

At test initiation, 250 mL of microbial inoculum was added to the first prepared flask to give a final volume of 500 mL.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Samples (2 x ca 5 L) of activated sewage sludge were obtained from Haddington Municipal Sewage Works (a local sewage processing plant, which handles predominantly domestic sewage) on 08 September 2015. On arrival at the laboratory approximately 500 mL of clear supernatant was removed from both samples and retained. Sewage sludge was maintained under aerated conditions for 2 days before use and fed daily with synthetic sewage feed at a rate of 50 mL/L.

The solid content of the sludge was determined by removing duplicate sub-samples (5 mL) of homogenised sludge (shaken vigorously to homogenise) and drying in an oven at approximately 105°C. The suspended solids content was higher than the required range of 3 g/L ± 10% therefore the solid content was adjusted by dilution until it reached 3.26 g/L which was within the acceptable range.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Not applicable
Test temperature:
18-22°C
pH:
7.21-7.36
Dissolved oxygen:
Measured during the study.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal dipentaerythritol concentrations: 10, 100 and 1000 mg/L
Nominal reference item: 5, 15 and 30 mg 3, 5-DCP/L
Details on test conditions:
The test was conducted in a temperature controlled laboratory set at a temperature in the range 18-22°C. Test solutions within the laboratory were maintained at 20 ± 2°C by placing the test vessels in a water bath. The temperature in the water bath was measured using a Brannan England digital in/out thermometer.

After preparation, the pH of each vessel was measured using a Mettler Toledo SevenGo Duo SG 23 pH meter. No pH adjustment was necessary.

Test vessels were aerated for 3 hours at a flow rate of 1.0 L/min. The flow rate of the air was measured using a Cache Quality Instrumentation Influx flow meter (range 0.1-1.2 L/min).

After the 3 h contact time (within 1 min) the contents of the first control vessel was poured to overflowing into a glass biological oxygen demand (BOD) bottle (250 mL capacity). The dissolved oxygen concentration was recorded at 30 second intervals for a period of 10 min. The above process was repeated for each vessel after the 3 hour contact time.

The dissolved oxygen concentration was measured using a YSI Incorporated, Dissolved Oxygen meter (Model No. 5100) and BOD probe (Model No. 5010) with agitator paddle.
Reference substance (positive control):
yes
Remarks:
3, 5-dichlorophenol (3, 5-DCP)

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The results demonstrated that there was no significant inhibition at 10, 100, or 1000 mg/L dipentaerythritol concentrations under the conditions of the test.

The 3h EC50 for dipentaerythritol to activated sludge was estimated to be to be greater than 1000 mg/L.

The 3h NOEC for dipentaerythritol to activated sludge was estimated to be 1000 mg/L.

Results with reference substance (positive control):
The 3h EC50 for 3,5-dichlorophenol to activated sludge was calculated to be 6.91 mg/L with 95% confidence limits of 5.94 and 8.04 mg/L, under the conditions of this test.
Reported statistics and error estimates:
The respiration rate for each vessel was calculated from the measured values of recorded oxygen concentrations versus time and expressed as mg O2/L/h for the linear part of the graphs, limiting the calculations of oxygen concentrations between approximately 7.0 and 2.0 mg O2/L. Where respiration was low (15 and 30 mg/L reference item vessels) calculations were taken for the full 10 minute oxygen measurement period when on the linear range.
The percentage inhibition of total oxygen consumption at each dipentaerythritol concentration was calculated from the respiration rate expressed as a percentage of the mean control respiration rate:
IT = [1-(RT/RTB)] x 100

Where:

RT = Total respiration rate at each tested concentration of test item
RTB = Mean respiration of control vessels
The percentage inhibition was calculated at each test concentration as above. No statistical analysis was conducted for dipentaerythritol as there was no inhibition.

Percentage inhibition was calculated for each of the 3, 5-DCP (reference item) concentrations.

The EC50 and associated 95% confidence interval for 3, 5-DCP was estimated using the non-parametric Trimmed Spearman-Karber Method (Hamilton et al, 1977).

Any other information on results incl. tables

Table 1: Respiration Rates and Percentage Inhibition

 

Treatment

Nominal Concentration (mg/L)

Replicate Flask

Respiration Rate (mg O2/L/h)

% Inhibition

Control

NA

I

52.20*

NA

II

43.75

NA

Mean

47.98

NA

Dipentaerythritol

10

I

52.80

0.0**

100

I

53.76

0.0**

1000

I

51.15

0.0**

II

48.76

0.0**

III

51.38

0.0**

Mean

50.43

0.0**

3, 5-DCP

5

I

30.98

35.4

15

I

6.69

86.1

30

I

3.73

92.2

Coefficient of Variation in Control Replicates (%)

12.6

NA – Not applicable

*Values converted into mg/L concentrations from % Saturated Dissolved Oxygen values, as % Saturated Dissolved Oxygen values recorded in error.

** Mean respiration rates in these treatments were higher than the mean of the control, giving rise to apparent negative inhibition values that are reported here as zero percent inhibition.

Table 2: Measured pH values in each vessel at test initiation

 

Treatment

Nominal Concentration (mg/L)

Replicate

pH

Control

NA

I

7.36

II

7.21

Dipentaerythritol

10

I

7.31

100

I

7.27

1000

I

7.26

II

7.22

III

7.23

3, 5-DCP

5

I

7.22

15

I

7.22

30

I

7.25

Table 3: Dissolved Oxygen Measurements at 3 h Contact Time during the Test

 

Time (min)

Dissolved Oxygen Measurements at 3hr Contact Time (mg/L)

Control

Nominal Concentration of Dipentaerythritol (mg/L)

3, 5 DCP (mg/L)

Control

C1*

10

100

1000 (I)

1000 (II)

1000 (III)

5

15

30

C2

0.5

7.49

7.32

7.66

5.58

7.50

6.71

7.59

9.26

9.18

7.31

1.0

7.06

6.86

7.21

5.16

7.10

6.28

7.35

9.22

9.17

6.98

1.5

6.68

6.45

6.78

4.74

6.69

5.87

7.08

9.18

9.15

6.64

2.0

6.23

6.01

6.32

4.31

6.30

5.44

6.83

9.10

9.13

6.29

2.5

5.81

5.54

5.88

3.88

5.91

5.01

6.59

9.05

9.10

5.90

3.0

5.39

5.08

5.43

3.45

5.49

4.56

6.33

9.01

9.06

5.53

3.5

4.95

4.64

4.97

3.02

5.07

4.14

6.06

8.94

9.02

5.16

4.0

4.52

4.23

4.53

2.59

4.67

3.70

5.81

8.89

9.00

4.79

4.5

4.11

3.74

4.05

2.17

4.26

3.28

5.55

8.82

8.96

4.42

5.0

3.68

3.26

3.63

1.74

3.85

2.84

5.29

8.78

8.93

4.07

5.5

3.23

2.74

3.18

1.35

3.44

2.42

5.03

8.71

8.90

3.72

6.0

2.81

2.45

2.73

0.94

3.03

2.00

4.77

8.66

8.86

3.34

6.5

2.33

2.02

2.30

0.54

2.62

1.57

4.51

8.60

8.83

2.95

7.0

1.93

1.51

1.86

0.19

2.22

1.16

4.25

8.53

8.79

2.60

7.5

1.54

1.12

1.42

0.00

1.82

0.76

3.91

8.48

8.76

2.24

8.0

1.10

0.73

1.03

-

1.42

0.39

3.73

8.42

8.72

1.89

8.5

0.69

0.35

0.58

-

1.06

0.08

3.47

8.37

8.69

1.55

9.0

0.35

0.05

0.20

-

0.68

0.00

3.21

8.32

8.65

1.21

9.5

0.06

0.00

0.00

-

0.33

-

2.95

8.26

8.62

0.58

10.0

0.00

0.00

0.00

-

0.04

-

2.70

8.20

8.59

0.40

 *Values converted into mg/L concentrations from % Saturated Dissolved Oxygen values, as % Saturated Dissolved Oxygen values recorded in error.

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The results are considered valid since the following OECD Guideline criteria were met:

- The mean oxygen uptake rate in the control vessels was 29.44 mg/oxygen/g of activated sludge (dry weight of suspended solids)/h.
(Criterion: should be >20 mg/oxygen/g of activated sludge (dry weight of suspended solids)/h)

- The coefficient of variation of oxygen uptake rate in control replicates was 12.6% (Criterion: should be <30%)

- The EC50 (3 h) for the reference item was in the range 2 to 25 mg/L (6.91 mg/L).

The 3h EC50 for dipentaerythritol to activated sludge was estimated to be to be greater than 1000 mg/L.
The 3h NOEC for dipentaerythritol to activated sludge was estimated to be 1000 mg/L.
Executive summary:

This study was conducted to determine the effects of dipentaerythritol on sewage micro-organisms by measuring the respiration rate under defined conditions. The study was designed to be appropriate for submission to Regulatory Authorities using procedures outlined in OECD Guideline 209 (July 2010).

The test was conducted at nominal concentrations of dipentaerythritol: 10, 100 and 1000 mg/L. One replicate flask was prepared for the 10 and 100 mg/L test treatments and three replicate flasks for the 1000 mg/L. A reference item (3, 5 dichlorophenol) was tested as a positive control at the following nominal concentrations: 5, 15 and 30 mg/L. Duplicate control vessels were also tested.

The prepared vessels were aerated for 3 h, after which dissolved oxygen concentrations were measured at 30 sec intervals for up to a maximum period of 10 min. The respiration rate (mg O2/L/h) was calculated over the linear phase of oxygen consumption for each vessel (ca. 2.0 – 7.0 mg O2/L). In the 15 and 30 mg 3, 5 DCP /L replicates where respiration rate were low, the calculation was conducted over the full linear range. The respiration inhibition (%) was compared with the mean control vessel respiration rate, for both the reference and test items. The test met all the validity criteria for the OECD Guideline 209 (July 2010).

The 3 h EC50 for 3, 5-dichlorophenol to activated sludge was estimated to be 6.91 mg/L, under the conditions of the test.

There was no respiration inhibition at the nominal concentrations of 10, 100 and 1000 mg dipentaerythritol/L. The 3 h NOEC for dipentaerythritol to activated sludge was therefore estimated to be 1000 mg/L under the conditions of the test. The 3 h EC50 for dipentaerythritol to activated sludge was calculated to be greater than 1000 mg/L.