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Toxicity to microorganisms

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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: in accordance with guidelines

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Algea Fert Solid K+
- Substance type: fertilizer ( algal extract)
- Physical state: solid, black micro-flakes
- Lot/batch No.: 00680
- Expiration date of the lot/batch: March 2012
- Stability under test conditions: Stable under suitable storage conditions.
- Storage condition of test material: original tightly-closed containers in well- ventilated place.

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The test item was tested at five concentrations in a geometric series, namely 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L.

The test solutions were prepared as follows: prior to the start of the test, a stock solution of 500.0 mg/L was prepared by direct weighting into tap water (0.2505 g of test item into 500 mL of tap water). The obtained solution appeared dark-brown and it was manually mixed to achieve the totally solubilisation of test item.
The test concentrations were prepared by taking adequate aliquots of stock solution and diluting them into tap water water.
3,5-dichlorophenol was tested at three concentrations, 5.0, 15.0 and 50.0 mg/L, under the same test conditions.

A stock solution of 3,5-dichlorophenol at 0.5 g/L was prepared according to the description reported on OECD Guideline no. 209.: 0.5 g of 3,5-dichlorophenol were dissolved in 10 mL 1M NaOH and diluted to about 30 mL with purified water, then approximately 7 mL of 1N H2SO4 were added. This procedure was adapted to the weighed reference item (0.05 g/L instead 0.5 g/L), which was then diluted by a factor of 10. The final pH was 7.53 and did not need any adjustment.
The test concentrations were prepared by taking adequate aliquots of stock solution and diluting them in deionised water .
One replicate per concentration and two for the control were prepared. Before the beginning of the test, 16 mL synthetic wastewater were added to the test flasks to give a volume of 300 mL at each test concentration. Immediately prior to the start of the test, 200 mL of the activated sludge inoculum (4 g/L dry weight) was poured into test bottles. Two control bottles containing tap water and synthetic wastewater only, were prepared at the beginning and at the end of the test.

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
The study was performed using activated sludge collected from the recirculation tank of ALSI (Alto Lambro Servizi Idrici) wastewater treatment plant treating mainly domestic wastewater. Four litres of activated sludge were kept on September 22, 2010 and transferred to ChemService laboratory where the sample was immediately put under aeration and shaking.
The temperature of the inoculum was 22.5 °C.
The sludge was washed by centrifugation, the supernatant liquid phase decanted and the solid material re-suspended in tap water. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated as 7.46%.
Based on this ratio, calculated amounts of wet sludge were suspended in the test water to obtain a concentration equivalent to 4.0 g dry material per litre. The obtained suspension was held under constant aeration, room temperature and dark conditions for one day prior to the test initiation.
Moreover 50 mL per litre of a synthetic feeding prepared immediately before use was added to the sludge inoculum.
The pH of sludge inoculum used for the test was 7.12 and its temperature was 21.4 °C. The temperature during the holding period was measured twice a day using a digital thermometer. It was in the range 21.4 – 21.7 °C .

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Test temperature:
The temperature in the test media, measured using a digital thermometer in each test vessel, was in the range of 19.7 – 20.3°C.
pH:
At the end of the test period, the pH values were in the range of 7.80 – 8.00 .
Nominal and measured concentrations:
The test item concentrations were 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L.
Details on test conditions:
The test item was tested at five concentrations in a geometric series, namely 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L.
The test solutions were prepared as follows: prior to the start of the test, a stock solution of 500.0 mg/L was prepared by direct weighting into tap water (0.2505 g of test item into 500 mL of tap water). The obtained solution appeared dark-brown and it was manually mixed to achieve the totally solubilisation of test item. The test concentrations were prepared by taking adequate aliquots of stock solution and diluting them into tap water.
3,5-dichlorophenol was tested at three concentrations, 5.0, 15.0 and 50.0 mg/L, under the same test conditions.
A stock solution of 3,5-dichlorophenol at 0.5 g/L was prepared according to the description reported on OECD Guideline no. 209.: 0.5 g of 3,5-dichlorophenol were dissolved in 10 mL 1M NaOH and diluted to about 30 mL with purified water, then approximately 7 mL of 1N H2SO4 were added. This procedure was adapted to the weighed reference item (0.05 g/L instead 0.5 g/L), which was then diluted by a factor of 10. The final pH was 7.53 and did not need any adjustment.
The test concentrations were prepared by taking adequate aliquots of stock solution and diluting them in deionised water . One replicate per concentration and two for the control were prepared.
Before the beginning of the test, 16 mL synthetic wastewater were added to the test flasks to give a volume of 300 mL at each test concentration. Immediately prior to the start of the test, 200 mL of the activated sludge inoculum (4 g/L dry weight) was poured into test bottles. Two control bottles containing tap water and synthetic wastewater only, were prepared at the beginning and at the end of the test. During the 3-hour incubation period, the contents of the test flasks were continuously aerated with compressed air (approximately 0.5 - 1 L/minute).
The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period. Prior to the measurement of the respiratory rate, the oxygen concentration always exceeded 6.5 mg/L.
The temperature in the test media, measured using a digital thermometer in each test vessel, was in the range of 19.7 – 20.3.
The respiration rate was measured after exactly three hours of incubation with an oxygen electrode and was recorded every minute for ten minutes. The oxygen consumption (mg O2 /L/minute) was determined from the linear part of the respiration curve (approximately 6.5 – 2.5 mg/L). During measurement, the sample was stirred continuously by a magnetic stirrer.
Water quality criteria: the pH was determined in the freshly prepared inoculum of sludge, in all the test concentrations and in the controls at the end of the test. Before the addition of activated sludge, the test media appeared as a suspension.


Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 100 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:
EC50
Effect conc.:
> 100 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:
other: EC80
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
The reference item, 3,5-dichlorophenol, was tested in parallel at three concentrations under the same test conditions to check the response of the sludge activate batch.
The 3-hour EC50 of the reference item (3,5-dichlorophenol) was 11.79 mg/L (within the range 5-30 mg/L).
Reported statistics and error estimates:
The statistical analysis was carried out using CETIS elaboration software v1.026D

Any other information on results incl. tables

Oxygen consumption by activated sludge in presence of different concentrations of Algea Fert Solid K+

Test item concentration

(mg/L)

Oxygen concentration (mg/L)

T0

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

0 (control)

6.38

5.63

4.83

3.89

3.11

2.52

n.d.

n.d.

n.d.

n.d.

n.d.

10.0

6.54

5.61

4.84

3.98

3.37

2.68

n.d.

n.d.

n.d.

n.d.

n.d.

17.8

6.38

5.47

4.62

4.24

3.29

2.65

n.d.

n.d.

n.d.

n.d.

n.d.

31.6

6.42

5.64

4.77

4.12

3.62

2.71

n.d.

n.d.

n.d.

n.d.

n.d.

56.2

6.66

5.72

4.88

4.41

3.54

2.87

2.61

n.d.

n.d.

n.d.

n.d.

100.0

6.72

5.78

5.14

4.27

3.72

2.96

2.63

n.d.

n.d.

n.d.

n.d.

0 (control)

6.64

5.79

5.15

4.49

3.66

2.89

2.51

n.d.

n.d.

n.d.

n.d.

n.d.: not determined as the values dropped below the 2.5 mg O2/L value.

Oxygen consumption by activated sludge in presence of different concentrations of 3,5-dichlorophenol

Reference item concentration

(mg/L)

Oxygen concentration (mg/L)

T0

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

5.0

6.53

5.69

5.21

4.48

3.72

3.12

2.85

n.d.

n.d.

n.d.

n.d.

15.0

6.47

5.24

4.75

4.37

4.18

3.94

3.78

3.58

3.22

3.08

2.53

50.0

6.61

5.73

5.61

5.42

5.27

5.18

5.07

4.96

4.87

4.68

4.19

n.d.: not determined as the values dropped below the 2.5 mg O2/L value.

Respiratory rates for the test concentrations and for the controls and active sludge respiration inhibition caused by test item after 3-h exposure

Test item concentration

(mg/L)

Rate of

respiration

(mg O2/L/h)

Respiration inhibition

(%) *

0 (control)

46.3

----

10.0

46.3

-5.7

17.8

44.8

-2.2

31.6

44.5

-1.6

56.2

45.5

-3.8

100.0

45.1

-3.0

0 (control)

41.3

----

* negative values indicate that a respiration greater than the mean respiration of two controls occurred, as provided by biological variability

Respiratory rates for the reference item concentrations and active sludge respiration inhibition in comparison to controls after 3-h exposure

Referenceitemconcentration

(mg/L)

Rate of

respiration

(mg O2/L/h)

Respiration inhibition

(%)

5.0

40.9

6.6

15.0

12.9

70.6

50.0

7.9

82.0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The test meets the validity criteria in that the respiration rate of the two controls differed by 10.8% (within the maximum difference of 15%) and the 3-hour EC50 of the reference item (3,5-dichlorophenol) was 11.79 mg/L .
Conclusions:
The toxic effect on aerobic treatment plant microorganisms of the test item Algea Fert Solid K+ was tested with activated sludge after 3-hours contact.
The endpoints after 3-hours exposure were determined to be as follows:

EC20 > 100 mg/L
EC50 > 100 mg/L
EC80 > 100 mg/L
Executive summary:

The toxicity of the test item Algea Fert Solid K+ to activated sludge was assessed in a respiration inhibition study according to:

- EU Commission Directive EC 440/2008, C.11.

- OECD Guideline No. 209, “Activated Sludge, Respiration Inhibition Test”, 1984.

 For this purpose, aerobic activated sludge collected from the recirculation tank of a municipal wastewater treatment plant was incubated for 3-hours with different concentrations of the test item under defined conditions.

 The test item concentrations were 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L. In addition to the five test item concentrations, two untreated controls were incubated under the same conditions.

 In order to check the normal sensitivity of the batch of activate sludge, 3,5-dichlorophenol was used as a reference item. For this purpose three nominal concentrations of 3,5-dichlorophenol were tested, 5.0, 15.0 and 50.0 mg/L respectively.

 The EC50of 3,5-dichlorophenol was found to be 11.79 mg/L, thus complying with the validity range indicated by the referenced guidelines (EC50: 5-30 mg/L).

The respiratory rate expressed as oxygen consumption was measured after three hours of exposure using a portable oximeter.

 The inhibitory effect of the test item is expressed as a percent of the mean respiratory rate of two controls. The results are related to the nominal concentrations of the test item.

 The respiration rate of the two controls was found to differ by 10.8%, thus complying with the validity criteria indicated by the mentioned guidelines (maximum difference between two replicates of control = 15%).

Based on the results, the EC20, EC50and the EC80for the test item were assessed to be greater than the highest tested concentration, i.e. greater than 100 mg/L.

At the start of the test the pH of test water was 7.43. The pH values and temperature of test solutions were measured in each test flask during the oxygen determination: the pH was in the range 7.80 – 8.00, while the temperature was found to be in the range 19.7 – 20.3°C, i.e. within the specified range (20.0± 2 °C).