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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Aug. 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was conducted according to an internationally recognised method, and under GLP. The substance is adequately characterised with its purity. A Klimisch score of 2 was however assigned due to a deviation from the validity criteria recommended in the current version of OECD 209 guideline. Indeed, two out of the three validity criteria were fulfilled; the third one being not in place in the 1984 version of the guideline that was used at the time of the study (see the field "Details on results" for further information). This deviation was not considered to have affected the results of the study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
OECD, 1984
Deviations:
no
Remarks:
The report mentions no deviation from the version of the guideline in place at that time, but some deviations from the study plan (see in the field "Any other information on materials and methods incl. tables").
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTIONS
- Method: A stock solution was prepared at test initiation by dissolving 265 mg ETFBO in 530 mL isotonic medium resulting in a concentration of 500 mg/L. The test solutions were prepared from this stock solution as reported in the table "Preparation of the test solutions" (see in the field "Any other information on materials and methods incl. tables").
- Controls: Isotonic medium + Synthetic waswater + Activated sludge (see the table "Preparation of the test solutions" in the field "Any other information on materials and methods incl. tables").
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No undissolved test substance was visible.
- Other relevant information: The stock solution was clear.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: No.
- Name and location of sewage treatment plant where inoculum was collected: About 5 L secondary (biological) activated sludge was obtained from the RWZI Horstermeer in Nederhorst den Berg, The Netherlands on 9 August 2007. The RWZI Horstermeer is an activated sludge plant treating 160,000 i.e. wastewater (about 80% domestic wastewater). The sludge was obtained from the second tank (from the building) on the day of test initiation and was used directly.
- Preparation of inoculum for exposure: After receipt at the laboratory, tubes with 200 mL of the activated sludge were centrifuged at 4000 rpm for 20 minutes. After centrifugation, one tube was used to determine the dry weight of the activated sludge. The activated sludge was dried for 2 hours at 105°C. The dry weight of the activated sludge appeared to be 1.75 g per 200 mL. The supernatant of the tubes was decanted and isotonic medium was added to a dry weight of 4.0 g/L. The minimum volume needed for the study was 2200 mL. The contents of 7 vessels (total dry weight of 12.25 g) was resuspended in 3100 mL isotonic medium resulting in a dry weight content of about 4 g/l.
No further data.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
Respiration rates were measured after 30 min and at the end of the 3-hour exposure period.
Hardness:
No data
Test temperature:
19.6 to 20.6°C; which was in agreement with the criterion of 20 +/- 1°C.
pH:
The pH of the synthetic waste water was 7.3; which was in agreement with the criterion of 7.5 +/- 0.5.
No data on the pH in the test beakers.
Nominal and measured concentrations:
Nominal concentrations: 0, 5, 10, 20, 40, 80, 160 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers .
- Fill volume: 1 L.
- Volume of solution: 500 mL.
- Aeration: Each test solution was aerated immediately and vigorously using filtered air (via carbon filter) after the addition of activated sludge.
- No. of vessels per concentration (replicates): 1 per ETFBO concentration and 1 per 3,5-dichlorophenol concentration.
- No. of vessels per control (replicates): 2.
- Sludge concentration (weight of dry solids per volume): 1.6 g/L dry weight content in the test solutions.
- Nutrients provided for bacteria: One litre synthetic waste water was prepared to provide the activated sludge with “feed” during the test. The synthetic waste water was prepared in purified water one day before the start of the test as follows:
* Peptone: 16 g/L.
* Meat extract: 11 g/L.
* Urea: 3 g/L.
* NaCl: 0.70 g/L.
* CaCl2.2H2O: 0.40 g/L.
* MgSO4.7H2O: 0.20 g/L.
* K2HPO4: 2.8 g/L.
- Nitrification inhibitor used: None, nitrification was not investigated in this study .
No further data.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Isotonic medium was prepared in purified water with a composition based on ISO 8192 (ISO, 1986):
* NaCl: 5 g/L.
* MgSO4.7H2O: 0.12 g/L.
No further data.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
No further data.

EFFECT PARAMETERS MEASURED
- Thirty minutes after the start of the incubation phase, the measurement phase was started. A 250-mL Erlenmeyer flask was completely filled with the test solution and stoppered without a headspace. Each minute the oxygen content was recorded (Hach HQ10 oxygen meter). After 11 minutes, the test solution was poured back into the glass beaker and incubation (with vigorous aeration) was continued. After three hours of incubation the oxygen content was measured again during an 11-minutes period.
- For each glass beaker, the respiration rate was determined for both measurement periods (i.e. after 30 min and after 3 h). The respiration rate is defined as the slope of the linear part of the curve and is expressed as mg oxygen per litre per minute. For each concentration the inhibition percentage of the respiration rate was calculated.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.
- Range finding study: No data.
No further data.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Results with test substance: The inhibition percentages of the respiration rate are presented in the table reported in the field "Any other information on results incl. tables". At a nominal ETFBO concentration of 5 mg/L, an inhibition of the respiration rate of 18% was observed after an incubation period of 30 minutes. However, this observation is not considered relevant as it was not observed after an incubation period of 3 hours. At the highest test concentration of 160 mg/L, the respiration inhibition was 37 and 35%, respectively after 30 minutes and 3 hours incubation. The E(I)C50 value after 30 minutes and 3 hours of incubation was thus > 160 mg/L. No NOEC was available in the report. In the absence of details on the statistical results, it was not possible to determine the highest concentration without significant effect on respiration rate.

- Validity criteria: The study report mentions two validity criteria. The first one being that the 3h-E(I)C50 of 3,5-dichlorophenol should be in the range of 5 to 30 mg/L; which was the case in the present study (3h-EC50 = 5.1 mg/L). The second one being that the difference in respiration rate of the two control replicates should be less than 15%; which was also the case in the present study (9.8% after 30 min and 2.8% after 3h). These two validity criteria were those in place in the 1984 version of OECD 209 guideline that was used at the time of the study. The current version of OECD 209 guideline mentions these two validity criteria, with different thresholds however, and also a third validity criterion:
* The 3h-EC50 of 3,5-dichlorophenol should be in the range of 2 to 25 mg/L (instead of 5 to 30 mg/L in the 1984 version); meaning this validity criterion is still fulfilled considering the 3h-EC50 value of 5.1 mg/L obtained during the study.
* The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% (instead of 15% in the 1984 version) at the end of definitive test; meaning this validity criterion is still fulfilled considering the coefficient of variation of 2.8% recorded after 3 hours during the study.
* The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. This validity criterion was not mentioned in the study report because not in place in the version of OECD 209 guideline used at that time. To see whether this validity criterion was fulfilled, it was needed to recalculate the value starting from a blank controls oxygen uptake rate expressed in another unit (0.35 to 0.36 mg oxygen/L/min):
Knowing that the test solutions contained 1.6 g/L dry weight content of activated sludge:
0.35 to 0.36 mg oxygen/L/min is equivalent to:
0.35 to 0.36 / 1.6 x 60 = 13.13 to 13.50 mg oxygen/g/h; which is thus below the requirement of 20 mg oxygen/g/h.
By discussing with the study director having managed this test, it was concluded this "lower sludge activity" did not impact the result of the study since the inhibitory effect was measured relative to the controls, and in addition, all the criteria in place in the 1984 version of the guideline have been fulfilled. The study was judged to be still valid to be used in the registration dossier, but a Klimisch reliability score of 2 (reliable with restriction) was assigned.
Results with reference substance (positive control):
- Results with reference substance valid? Yes (3h-EC50 of 3,5 -dichlorophenol within the acceptance range which was reported to be 5-30 mg/L in the study report and 2-25 mg/L in the current OECD 209 guideline).
- 3h-EC50 = 5.1 mg/L.
Reported statistics and error estimates:
Linear interpolation, with concentration on a logarithmic scale (base = 10), was used to determine the EC50 after 30 minutes and after 3 hours.

Respiration rates and inhibition percentages:

Treatment

Concentration (mg/L)

Respiration rate after 30 min (mg oxygen/L/min)

Inhibition after 30 min (%)

Respiration rate after 3 h (mg oxygen/L/min)

Inhibition after 3 h (%)

Control

0 (replicate A)

0.32

-

0.36

-

0 (replicate B)

0.29

-

0.35

-

3,5-dichlorophenol

5

0.14

54.1

0.18

49.3

10

0.10

67.2

0.11

69.0

20

0.05

83.6

0.06

83.1

ETFBO

5

0.25

18

0.38

-7.0

10

0.31

-1.6

0.38

-7.0

20

0.32

-4.9

0.37

-4.2

40

0.31

-1.6

0.32

9.9

80

0.27

12

0.34

4.2

160

0.19

37

0.23

35

Validity criteria fulfilled:
not specified
Remarks:
See the discussion on the validity criteria in the field "Details on results".
Conclusions:
Initial exposure to ETFBO and then to its hydrolysis product triggered less than 50 % effects on activated sludge respiration rate at the highest tested concentration; the 3h-EC50 was thus set superior to 160 mg/L. In the absence of details on the statistical results, it was not possible to determine the NOEC.
Executive summary:

The toxicity to activated sludge was investigated in a GPL-compliant study performed according to OECD test guideline 209.

Activated sludge of a predominantly domestic sewage were exposed for 3 hours to a control treatment, to ETFBO at concentrations of 5, 10, 20, 40, 80 and 160 mg/L and to a reference substance, 3,5-dichlorophenol, at concentrations of 5, 10 and 20 mg/L. Oxygen contents were measured after 30 minutes and 3 hours. The respiration rates were determined for both measurement periods and the inhibition percentage of the respiration rates were calculated.

For 3,5-dichlorophenol, inhibition percentages of 49.3, 69.0 and 83.1 % were observed at the concentrations of 5, 10 and 20 mg/L, respectively. The 3h-EC50 for this reference substance was established to be 5.1 mg/L. For ETFBO, at the highest test concentration of 160 mg/L after 3 hours, the respiration inhibition was 35 %. Because less than 50 % effects were observed at the highest tested concentration, the 3h-EC50 was set superior to 160 mg/L. In the absence of details on the statistical results, it was not possible to determine the NOEC.

Description of key information

Initial exposure to ETFBO and then to its hydrolysis product triggered less than 50 % effects on activated sludge respiration rate at the highest tested concentration; the 3h-EC50 was thus set superior to 160 mg/L. In the absence of details on the statistical results, it was not possible to determine the NOEC.

Key value for chemical safety assessment

Additional information

An experimental study, conducted according to OECD guideline and GLP, is available. Despite a limitation due to the use of a version of OECD 209 guideline presenting different validity criteria as compared to the ones currently in place, the study is considered as reliable with restriction, and the result is retained as key data.