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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
UBA Guideline: "Bestimmung der akuten Bakterientoxizität, LTWS No 10, Sept. 79") and GLP; determination of Toxicity Threshold (TT) und "Wasserbewertungszahl" (WBZ
Qualifier:
according to guideline
Guideline:
other: UBA Guideline: "Bestimmung der akuten Bakterientoxizität, LTWS No 10, Sept. 79
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A test substance solution of 136000 mg/L Thiourea/L in sterile Milli-Q- water (Millipore Corp., Bedford, Mass., USA) was prepared. The test substance solution was diluted 1.25* in sterile Milli-Q water.
- The pH of the test substance solution was 6.9.
- Four parallel dilution series in 300 mL Erlenmeyer flasks stoppered with aluminium caps were prepared from the prepared test substance solution described above and sterile Milli-Q water.
- Each of the dilutions contained 1 part v/v of the test substance solution in 2^0 to 2^11 parts v/v mixture. The dilution series were prepared as follows: the first flask of each series contained 160 ml of test substance solution, from this flask the subsequent dilution steps at a constant dilution ratio were prepared by consistently mixing 80 ml of preliminary test substance dilution and 80 mL sterile Milli-Q water. Consequently, each flask contained 80 mL of test substance solution at the start.
- Each flask of the three inoculated dilution series (I, II, III) was made up to a final volume of 100 mL by adding 5 ml of a stock solution I, 5 mL of a stock solution II (see field "Any other information on materials and methods incl. tables" for composition of stock solutions) and 10 mL of the prepared bacterial suspension (see field "Details on inoculum") from the preliminary culture having a known adjusted extinction value.
- Following inoculation, the extinction value at 436 nm will correspond to the extinction value of the Formazin standard suspension TU/F/436 nm = 10.
- The flasks of the dilution series that were not inoculated (series t), were made up to 100 mL by adding 5 mL of stock solution I, 5 mL of stock solution II and 10 mL of saline.
- Ten control culture flasks (series B) with 80 mL sterile Milli-Q water, 5 mL stock solution I, 5 mL stock solution II and 10 mL prepared bacterial solution were also inoculated.
- Both inoculated and non-inoculated dilution series as well as the control flasks were left at 25 °C for 18 +/- 2 hours.
- After termination of the test period, colouration or turbidity occurred in the dilution series for chemical-physical reasons, the analogous steps of dilution of the non-inoculated series will be used as photometric blank values for turbidity of the inoculated dilution series.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
STOCK CULTURES OF PSEUDOMONAS PUTIDA:
Stock cultures of Pseudomonas putide, obtained from RIVM, Bilthoven, The Netherlands, were kept on thenutrient used for stock and preliminary cultures in agar plant tubes. New stock cultures were started at intervals of 1 week. The inoculated stock cultures were incubated at 25°C.

PRELIMINARY CULTURES OF PSEUDOMONAS PUTIDA:
Using aseptic techniques, small amounts of bacteria from a 7-day old stock culture of Pseudomonas putida were inoculated in fluid nutrient medium in Erlenmeyer flasks. The preliminary cultures were incubated at 25 °C for 18 +/- 2 hours. Subsequently the extinction of the monochromatic radiation at 436 nm for a 10 mm layer of bacterial suspension was determined by photoelectric measurement. On the basis of the values measured, the final turbidity value of the bacterial suspension was adjusted by means of sterile saline in such a way that the extinction value for a sample that has been subject to onward dilution 1 + 9 with saline correspond with the extinction value of a Formazin standard suspension TU/F/436 nm = 10. These preliminary cultures of bacterial suspension were used for inoculation of the test flasks.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
18 h
Remarks on exposure duration:
+/- 2 hours
Post exposure observation period:
Not applicable.
Hardness:
no data
Test temperature:
25 °C
pH:
6.9
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Concentration range, nominal: 53.1 to 108800 mg/L (corresponding to the following dilution series: 2^0, 2^1, 2^2, 2^3, 2^4, 2^5, 2^6, 2^7, 2^8, 2^9, 2^10, 2^11)
Details on test conditions:
TEST SYSTEM
- No. of vessels per concentration: 3
- No. of vessels per control: 1

EFFECT PARAMETERS MEASURED:
- concentration of bacterial suspension (measured turbidimetrically; expressed as the extinction of the primary light of the monochromatic radiation at 436 nm for a ölayer thickness of 10 mm)
- the toxicity threshold (TT) was determined graphically
Reference substance (positive control):
yes
Remarks:
methanol
Key result
Duration:
18 h
Dose descriptor:
other: toxicity threshold
Effect conc.:
1 265.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell multiplication inhibition
Details on results:
Based on the TT the "Bewertungszahl" (Assessment figure or value for bacteriotoxicity) is 2.9 (Bewertung wassergefährdender Stoffe, 1979).
Results with reference substance (positive control):
- Results with reference substance valid? yes (With respect to the known literture TT value of methanol (6600 mg/L) and considering the existing differences in definitions for the TT value in the literature concerned, it can be concluded that the test conditions were optimal and the results obtained are valid).
- Relevant effect levels: TT value (90 %-line) = 13043 mg/L
Reported statistics and error estimates:
Calculations and interpretation of results:
The following values were calculated:
- mean extinction value of the control cultures (series B)
- mean extinction value of each concentration of the inoculated dilution series (series I, II, III)
- mean extinction value of the control cultures, reduces by the 10 % extinction-difference value
The mean extinbction values of each dilution step were plotted against the logarithm of the mean values of the concentration of the test substance. The mean value of the control culture, reduced by the 10 % extinction-difference value, was plotted as a horizontal reference

None of the concentrations in the dilution series of thiourea showed colouration or turbidity; therefore, no correction for the extinction value of the dilution series was made.

Table 1: Extinction values of the inoculated dilution series I, II, II, the control flaks B and reference dilution series R

Extinction 436 nm

 

Methanol

Thiourea

Mean

B

mg/L

R

mg/L

I

II

III

I, II, III

.750

4937.5

.610

53.1

.700

.718

.715

.711

.725

9875.0

.640

106.2

.705

.728

.702

.712

.708

19750.0

.649

212.5

.690

.721

.687

.699

.749

39500.0

.413

425.0

.680

.673

.640

.664

.710

79000.0

.087

850.0

.720

.712

.708

.713

.740

 

 

1700.0

.605

.612

591

.603

.730

 

 

3400.0

.665

.620

.582

.622

.727

 

 

6800.0

.326

.415

.300

.347

.749

 

 

13600.0

.071

.060

.056

.062

.737

 

 

27200.0

.047

.046

.046

.046

 

 

 

54400.0

.060

.067

.064

.064

 

 

 

108800.0

.067

.065

.078

.070

Mean:

0.733

 

 

 

 

 

 

 

Methanol reference

Thiourea

90 %-line

TT value

TT value

.659

13043 mg/L

1265.4 mg/L
Validity criteria fulfilled:
yes
Conclusions:
The toxicity thresholf (TT) value of thiourea for Pseudomonas putida bacteria is 1265.4 mg/L.
Executive summary:

Thiourea was investigated for its ability to inhibit the cell multiplication of the bacteria species Pseudomonas putida according to the Guideline "Bestimmung der akuten Bakterientoxizität, LTWS No 10, Sept. 79" of the German Environmental Protection Agency (UBA).


 


Cultures of the bacteria were exposed to nominal concentrations ranging from 53.1 to 108800 mg Thiourea/L for 18 +/- 2 hours.


 


Based on the solubility of Thiourea in water, a toxicity threshold (TT) value of Thiourea of 1265.4 mg/L for Pseudomonas putida could be determined. The “Bewertungszahl” (Assessment figure or value for bacteriotoxicity) is therefore 2.9 (Bewertung wassergefährdender Stoffe, 1979).

Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Remarks:
cited in the BUA-Stoffbericht. The original reference from Downing et al. (1964) in J. Inst. Sew. Purif. 537-554 (1964) was not available for review.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Inhibition of nitrification due to thiourea was elucidated with a "fill and draw" laboratory equipment.
GLP compliance:
not specified
Analytical monitoring:
not specified
Details on sampling:
no data
Vehicle:
not specified
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Initial biomass concentration: 1.35 to 1.7 g/L
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
2.5 h
Remarks on exposure duration:
In the 2.5 h short-term study the inhibiting effect of thiourea on non-adapted domestic sewgae sludge microorganisms was determined. In addition a long-term nitrification inhibition experiment with increasing concentrations was conducted over 110 days.
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
short term experiment: 25 °C
long term experiment: 20 °C
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
short-term experiment: 0.076 mg/L thiourea
long-term experiment: 0.06 to 92 mg/L thiourea
Details on test conditions:
no data
Key result
Duration:
2.5 h
Dose descriptor:
other: IC75
Effect conc.:
0.076 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Details on results:
no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data

The long-term experiment could demonstrate that the inhibition of the nitrifying capability of the domestic sewage sludge was negligible. No inhibition of nitrification could be observed in the first six days of the experiment at a thiourea concentration of 0,06 mg/L.

Within the next 26 days thiourea concentration was increased to 1 mg/L which did also not result in ntirification inhibition.

Subsequent sudden raised concentrations of thiourea in the influent led to considerably raised NH4 levels in the effluent. Over the next days thiourea concentrations were partially further increased a lower nitrification rate could be measured until full adaption of the activated sludge microorganisms.

Validity criteria fulfilled:
not specified
Conclusions:
Nitrification in non-adapted activated sludge is inhibited by a thiourea concentration of 0.076 mg/L to a degree of 75% (EC75 = 0.076 mg/L). Adaption to thiourea could be shown in a long-term experiment.
Executive summary:

In a 2.5 h short-term experiment the inhibiting effect of thiourea on non-adapted domestic sewgae sludge microorganisms was determined. Nitrification in unadapted activated sludge is inhibited by a thiourea concentration of 0.076 mg/l to a degree of 75%.


 


In a following long-term experiment over 110 days adaption of the microorganisms to thiourea added in a concentration range of 0.06 mg/L to 92 mg/l could be shown:


- No inhibition of nitrification could be observed in the first six days of the experiment at a thiourea concentration of 0,06 mg/L.


- Within the next 26 days thiourea concentration was increased to 1 mg/L which did also not result in ntirification inhibition.


- Subsequent sudden raised concentrations of thiourea in the influent led to considerably raised NH4 levels in the effluent. Over the next days thiourea concentrations were partially further increased a lower nitrification rate could be measured until full adaption of the activated sludge microorganisms.

Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Remarks:
cited in the BUA-Stoffbericht. The original reference was not available for review.
Qualifier:
according to guideline
Guideline:
ISO 9509 (Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms)
Version / remarks:
modified
Principles of method if other than guideline:
no data on principles
GLP compliance:
not specified
Analytical monitoring:
not specified
Details on sampling:
no data
Vehicle:
not specified
Details on test solutions:
no data
Test organisms (species):
activated sludge, domestic
Details on inoculum:
communal activated sludge
- Initial biomass concentration: 1.4 g suspended solids/L; pH 7.61
Test type:
not specified
Water media type:
not specified
Limit test:
no
Remarks on exposure duration:
no data
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
no data
pH:
7.61
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
no data
Details on test conditions:
no data
Reference substance (positive control):
not specified
Duration:
2.5 h
Dose descriptor:
other: EC20
Effect conc.:
0.19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Key result
Duration:
2.5 h
Dose descriptor:
EC50
Effect conc.:
0.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Details on results:
no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data

no data

Validity criteria fulfilled:
not specified
Conclusions:
An EC20 of 0.19 mg/L and an EC50 of 0.35 mg/L of thiourea could be identified.
Executive summary:

A nitrification inhibition test was conducted according ISO 9509 (modified) with thiourea and communal activated sludge (1.4 g SS/L; pH 7.61). An EC20 of 0.19 mg/L and an EC50 of 0.35 mg/L could be identified.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
other: DIN 38412 part 24
Principles of method if other than guideline:
Characterisation of the influence of thiourea on aerobic biological process like nitrification according "DIN 38412 Teil 24"
GLP compliance:
not specified
Analytical monitoring:
not specified
Details on sampling:
no data
Vehicle:
not specified
Details on test solutions:
no data
Test organisms (species):
activated sludge, domestic
Details on inoculum:
pre-treated communal wastewater
- Pretreatment: yes
- Initial biomass concentration: 2 to 3.5 g/L
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
135 d
Remarks on exposure duration:
4.5 month
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
15 - 20.5 °C
pH:
7.15 - 8.07
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Concentrations of thiourea added: 0.04 to 3 mg/L
Details on test conditions:
- Biomass loading rate: 2 g/L and 3.5 g/L
Reference substance (positive control):
not specified
Duration:
10 min
Dose descriptor:
other: EC20
Effect conc.:
205 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Key result
Duration:
10 min
Dose descriptor:
EC50
Effect conc.:
4 500 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Duration:
10 min
Dose descriptor:
other: EC80
Effect conc.:
100 000 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Details on results:
no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data

Results experiment 1:

The BOD5 was not affected by addition of thiourea whereas nitrification was inhibited, even in adapted activated sludge.

The decomposition rate for organic substances in sewage effluent was determined to be 89.9%.

Results experiment 2:

Thiourea was added up to concentrations of 50 mg/l. Up to 12 mg/L nitrification was not inhibited. The decomposition rate for organic substances in sewage effluent was not negatively influenced up to the maximum concentration of 50 mg/L.

Thiourea was decomposed as in the effluent less substance than added was detectable. The rate of decomposition was determined to be 93.4 % (21 mg/l thiourea added) and 98 % (50 mg/l thiourea added), respectively.

Results Experiment 3: Thiourea does inhibit nitrification in non-adapted activated sludge.

Validity criteria fulfilled:
not specified
Conclusions:
Thiourea was shown to inhibit nitrification in non-adapted activated sludge.
Executive summary:

Several experiments were conducted to investigate the influence of thiourea on non-adapted, activated sludge:


 


The first experiment was conducted according to DIN 398412 part 24 for a total duration of 4.5 month. Thiourea was added in concentrations of 0.04 to 3 mg/L. The activity of the activated sludge was measured by determination of BOD5. The BOD5 was not affected by addition of thiourea whereas nitrification was inhibited, even in adapted activated sludge.


The decomposition rate for organic substances in sewage effluent was determined to be 89.9%.


 


In experiment 2 that immediately followed experiment 1, thiourea was added in concentrations up to 50 mg/L. Up to 12 mg/L nitrification was not inhibited. The decomposition rate for organic substances in sewage effluent was not negatively influenced up to the maximum concentration of 50 mg/L.


Thiourea was decomposed as in the effluent less substance than added was detectable. The rate of decomposition was determined to be 93.4 % (21 mg/L thiourea added) and 98 % (50 mg/L thiourea added), respectively.


 


In a third experiment the short-term respiration inhibition (10 min) of thiourea on domestic activated sludge (1g/L) was determined. The EC50 (10 min) was determined to be 4500 mg/L.


 

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Remarks:
cited in the BUA-Stoffbericht. The original reference - Malaney et al. (1967) - was not available for review.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Determination of the oxygen consumption using a Warburg respirometer (respiration inhibition test).
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
activated sludge, domestic
Details on inoculum:
The initial concentration of the inoculum was 2.5 g/L. In a further study the concentration of the inoculum was increased to 5 g/L.
Test type:
not specified
Water media type:
freshwater
Total exposure duration:
144 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
20°C
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Thiourea was added at a concentration of 500 mg/L.
Details on test conditions:
Warburg respirometer
Reference substance (positive control):
not specified
Duration:
144 h
Dose descriptor:
EC100
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: A thiourea concentration of 500 mg/L led to inhibition of respiration in two experiments.
Key result
Duration:
144 h
Dose descriptor:
other: EC95
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: In a third experiment a very low oxygen consumption of 5.7 % could be determined.
Details on results:
A thiourea concentration of 500 mg/L led to inhibition of respiration in two experiments. In a third experiment a very low oxygen consumption of 5.7 % from theoretical could be determined.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data

The concentration of the inoculum was raised from 2.5 g/L to 5 g/L. This did not result in any changes.

Validity criteria fulfilled:
not specified
Conclusions:
Using the Warburg technique, a concentration of 500 mg/L thiourea was found to be toxic or very poorly oxidized by various activated sludges.
Executive summary:

The oxygen consumption of activated sludge was determined after addition of 500 mg thiourea/L in a Warburg respirometer. Thiourea was shown to completely inhibit respiration at a concentration of 500 mg/L in two experiments. In a third experiment a oxygen consumption of 5.7 % was recorded.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Remarks:
the publication is cited in the BUA-Stoffbericht. The original reference "NAPM National Association of Photographic Manufacturers: Environmental effect of photoprocessing chemicals (1987)" was not available for review.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The inhibitory effect of thiourea in concentrations 100 and 1000 mg/L on activated sludge was determined by measuring the respiration rate in a Warburg repirometer
GLP compliance:
not specified
Specific details on test material used for the study:
no data
Analytical monitoring:
not specified
Details on sampling:
no data
Vehicle:
not specified
Details on test solutions:
no data
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Activated sludge, non-adapted (1g/L) and adapted (2 g/L).
Test type:
not specified
Water media type:
not specified
Limit test:
no
Remarks on exposure duration:
no data
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
no data
pH:
6.5 - 7.5
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
100 and 1000 mg/L thiourea were added (nominal concentration)
Details on test conditions:
The oxygen uptake was determined using a Warburg respirometer.
Reference substance (positive control):
not specified
Key result
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: in the first 400 min inhibition was observed; at the end 100 mg/L thiourea did not have inhibitory effects on not-adapted acitvated sludge
Details on results:
1000 mg/L thiourea inhibited actived sludge strongly (in comparison to the control the oxygen uptake of non-adapted activated was determined to be 47.5%)
Exposure to 100 mg/L led to an adaption of the microorganisms in activated sludge after 400 min.
Adaption of the biomass to 200 mg/L thiourea during 7 days led to a decomposition of 276 mg/L thiourea of 32.5 % (referred to COD).
Results with reference substance (positive control):
no data

1000 mg/L thiourea inhibited oxygen uptake in comparison to the control by 47.5 %. No change in COD was measured.


100 mg/L induced inhibition of oxygen uptake during 400 min of the experiment. Afterwards oxygen uptake was comparable to the control (105%).


Adaption of the biomass to 200 mg/L thiourea during 7 days led to a decomposition of thiourea of 32.5 % (referred to COD).

Validity criteria fulfilled:
not specified
Conclusions:
Thiourea inhibits the activity of activated sludge. Microorganisms can adapt to thiourea.
Executive summary:

The effects of thiourea on sludge, either adapted or non-adapted, was investigated using a Warburg respirometer. It was shown that 1000 mg/L thiourea induce an inhibition of oxygen uptake in comparison to the control by 47.5 %. However, microorganisms can adapt to thiourea.


100 mg/l thiourea induced inhibition of oxygen uptake during 400 min of the experiment. Afterwards oxygen uptake was comparable to the control (105%).


Adaption of the biomass to 200 mg/L thiourea during 7 days led to a decomposition of thiourea of 32.5 % (referred to COD).

Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1966
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A method is described for determining the short term effects of sewages, effluents and individual substances on the nitrifying ability of activated sludge and the results of screening many substances are listed. The effects of mixtures of inhibitors and the possibility of formation of complexes between some of those inhibitors were investigated. The long term effects of inhibitors often differ from their immediate effects, one of the most important factors being the ability of activated sludge to become adapted to the inhibitor.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
After centrifugation of the liquor nitrate and nitrite were determined colorimetrically
Vehicle:
not specified
Details on test solutions:
Sewage was diluted with equal volume of water; BOD of 250 mg/L; 50-80 N/l ammonia
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge from a residential district of Stevenage. Mixed liquor from this plant was centrifuged to separate the activated sludge; the sludge was then washed once with distilled water, centrifuged and finally suspended in an equal weight of distilled water.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
2.5 h
Remarks on exposure duration:
a short-term test was conducted; the duration of the long-term test was reported to be 110 days
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
25°C
pH:
pH of the media: 7.6
Dissolved oxygen:
> 1mg/L O2
Salinity:
no data
Nominal and measured concentrations:
short term test: 0.076 mg/L Thiourea (10^-6 M)
long term test: 0.038 mg/L initial concentration; increasing concentration up to 0.76 mg/L; highest concentration tested 76 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical flask
- Material, size, headspace, fill volume: final volume of 100 mL
- Aeration: 12 mg/L O2/L per h
- Biomass loading rate: 1350 mg to 1700 mg dry matter/L

Long-term test: automatic fill-and draw activated sludge units
Reference substance (positive control):
not specified
Duration:
2 h
Dose descriptor:
other: EC75
Effect conc.:
0.076 mg/L
Nominal / measured:
nominal
Conc. based on:
other: amount of oxidized nitrogen from ammonia
Basis for effect:
inhibition of nitrification rate
Key result
Duration:
110 d
Dose descriptor:
EC0
Effect conc.:
>= 0.76 mg/L
Nominal / measured:
nominal
Conc. based on:
other: amount of oxidized nitrogen from ammonia
Basis for effect:
inhibition of nitrification rate
Details on results:
Acclimatisation of the microorganisms in the activated sludge was observed. The inhibitory effects on unacclimatised sludge are much greater than those on the sludges from the plants which had received thiourea for some time. Biological oxidation of thiourea to degradation products like nitrate and sulphate was detected by an increasing content of them in the effluent.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data
Validity criteria fulfilled:
not specified
Conclusions:
Thiourea was shown to have inhibitory effects on nitrification of unadapted microorganisms during short-term exposure. Long-term-exposure to increasing concentrations of thiourea leads to an acclimatisation of the microorganisms and decomposition of thiourea to sulphate and nitrate.
Executive summary:

The short-term and long-term effects of thiourea exposure to microorganisms in activated sludge were investigated. It could be shown that short term exposure to 0.076 mg/L thiourea leads to inhibition of 75 % of nitrification activity, whereas long-term exposure results in acclimatisation of microorganisms to thiourea up to concentration of 0.76 mg/L. Furthermore decomposition of thiourea could be observed by detecting the degradation products nitrate and sulphate in the effluent. A gradual increase of thiourea concentrations up to 76 mg/L showed an increasing oxidation of thiourea to the degradation products nitrate and sulphate.

Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1981
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effects of compounds potentially toxic to nitrification were examined using a laboratory procedure which involved aerating activated sludge mixed liquors in the presence of measured amounts of these compounds.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
The liquors were filtered.
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Thiourea was added in measured amounts to 300 ml of activated sludge.
Thiourea concentrations used: 1*10-8 M, 5*10-6 M, 10*10^-6 M
Test organisms (species):
activated sludge
Details on inoculum:
Activated sludge was aerated usually overnight, diluted with fresh settled sewage to give a liquor of 2500-3000 mg/solids. The liquor was filtered through a Whatman 2 V paper, the pH, ammonmia, nitrite and nitrate were determined immediately in the filtrate.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
2.5 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
20 - 25 °C
pH:
7.1-7.7
Dissolved oxygen:
30 % - 70 %
Salinity:
no data
Nominal and measured concentrations:
nominal concentration of thiourea: 1*10^-6, 5*10^-6 and 10*10^-6 M
Details on test conditions:
no data
Reference substance (positive control):
not specified
Duration:
2.5 h
Dose descriptor:
other: EC77
Effect conc.:
>= 0.76 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Ammonia loss
Basis for effect:
inhibition of nitrification rate
Key result
Duration:
2.5 h
Dose descriptor:
NOEC
Effect conc.:
0.38 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Ammonia loss
Basis for effect:
inhibition of nitrification rate
Details on results:
The inhibitory effects of the compounds variied from day to day according to the nitrifying capability of the sludge.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data

The results of different substances, among them thiourea, on their inhibitory effects on nitrification are descirbed in the study. A concentration of 0.076 mg/l (10^-6 M) and 0.38 mg/l (5*10^-6 M) thiourea does not lead to inhibition of nitrification wherease a concentration of 0.76 mg/l induces a 77% inhibition of nitrification in terms of ammonia loss measured.

Validity criteria fulfilled:
not specified
Conclusions:
Thiourea concentrations of 10*10-6 M led to 77 % inhibition of nitrification in terms of measured ammonia loss in the media.
Executive summary:

Inhibition of nitrification by thiourea concentrations of 1*10^-6, 5*10^-6 and 10*10^-6 M was measured in activated sludge by determination of ammonium loss and calculation of oxidized nitrogen production. Concentrations of 10*10^-6 M, corresponding to 0.76 mg/L thiourea led to an inhibition of nitrification of 77 %.


The results obtained for thiourea and its analogues provide further support for an hypothesis suggested as an explanation of the toxicity of compounds containing the N-C-S group; this is based on a model reaction involving copper in oxidases in Nitrosomonas.


It can be concluded that thiourea affects nitrification in an inhibitory manner at concentrations exceeding 0.76 mg/L.

Description of key information

Thiourea inhibits nitrification in non-adapted activated sludge. This is the most sensitive endpoint regarding metabolic processes in microorganisms. Sludge samples drawn on different days showed considerable variation in effects on nitrifying capability caused by thiourea (Wood et al., 1981). Similar variation was also observed in other studies. The inhibitory effect of thiourea varies depending on the composition of the microbial community in the activated sludge.
Short-term exposure of non-adapted activated sludge leads to inhibition of nitrification activity of up to 75 % at concentrations ranging between 0.76 mg/L and 0.076 mg/L. Although thiourea was shown to be a powerful inhibitor of nitrification in sewage sludge in short-term experiments, long-term experiments demonstrate that microorganisms quickly acclimatise to thiourea and nitrification continues in activated sludge.
According to the guidance on information requirement,s r7b, short-term measurements in the order of hours are preferred, based on the hydraulic retention time in a STP. The NOEC (2.5 h) of 0.38 mg/l with respect to inhibition of the nitrifying ability of STP microorganisms by thiourea (Wood et al., 1981) is used in the risk assessment. This value was chosen for the risk assessment in a weight of evidence approach (for details please refer to the discussion below).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
0.38 mg/L

Additional information

Several studies are available that describe the effects of thiourea on cell multiplication, nitrification or respiration rate of microorganisms. However, all data except for the study on cell multiplication inhibition of Pseudomonas putida have a low reliability (high Klimisch score) and are therefore used in a weight-of-evidence approach.

Coenen (1988) determined the toxicity threshold (TT) value of thiourea for the bacteria species Pseudomonas putida to be 1265.4 mg/l, corresponding to the “Bewertungszahl” (assessment figure or value for bacteriotoxicity) of 2.9 (Bewertung wassergefährdender Stoffe, 1979). Results for growth inhibition of Pseudomonas putida, however, are to be used for PNECstp derivation only if no other tests are available.

In a short-term experiment (2.5 h) Wood et al. (1981) measured inhibition of the nitrification rate to be 77 % at a thiourea concentration of 0.76 mg/L, and no inhibition at 0.076 and 0.38 mg/L. In contrast, Tomlinson et al. (1966) and Downing et al. (1964) could already observe 75 % inhibition of nitrification at concentrations as low as 0.076 mg/L after 2.5 h exposure.

In an additional long-term experiment (110 days) Tomlinson et al. (1966) could demonstrate acclimatisation of microorganisms to thiourea up to a concentration of 76 mg/L. Furthermore, degradation of thiourea could be observed by detecting the degradation products nitrate and sulphate in the effluent of a laboratory scale STP.

A nitrification inhibition test was conducted according to ISO 9509 (modified) with thiourea and communal activated sludge (1.4 g SS/L; pH 7.61). An EC20 of 0.19 mg/L and an EC50 of 0.35 mg/L could be determined (Forschungsvorhaben, 1991).

Besides, Xiong et al. (1998) could show that, once microorganisms were acclimatised to thiourea, the acquired tolerance persisted even after stopping the addition of thiourea for several weeks.

Grünwald (1984) conducted several experiments to investigate the influence of thiourea on respiration of non-adapted activated sludge. In a first experiment over 4.5 months (DIN 398412 part 24) thiourea was added at concentrations of 0.04 to 3 mg/l. The BOD5 was not affected by addition of thiourea whereas nitrification was inhibited even in adapted activated sludge. In a second experiment that immediately followed experiment 1, thiourea was added at concentrations up to 50 mg/L. Up to 12 mg/L nitrification was not inhibited.

In a third experiment the short-term respiration inhibition (10 min) of thiourea on domestic activated sludge (1g/l) was determined. The EC50 (10 min) was determined to be 4500 mg/L.

NAPM (1974) reports similar results. 1000 mg/l thiourea induced inhibition of oxygen uptake by 47.5 % in comparison to the control (Warburg respirometer).

By contrast, Malaney et al. (1967) measured complete respiration inhibition at a concentration of 500 mg/l in two experiments. In a third experiment oxygen consumption of 5.7 % compared to the control was recorded.

The above results clearly demonstrate that inhibition of nitrification by thiourea is the most sensitive endpoint for thiourea bacteriotoxicity. Thus, this endpoint is carried forward in the risk assessment.

The studies on nitrification inhibition conducted by Tomlinson et al. (1966) and Wood et al. (1981) are of higher quality compared to those by Downing et al. (1964), Forschungsvorhaben (1991), Xiong et al. (1998), and Grünwald (1984), as for these literature data only abstracts or secondary sources are available.

A comparison of the documentation, study setup and results from Tomlinson et al. (1966) and Wood et al. (1981) shows that both experiments were conducted in a similar way. The results are within the same order of magnitude. However, overall the experiment of Wood et al. (1981) is considered to be more elaborate due to analytical verification of test substance concentrations. In addition, Wood et al. (1981) used several test substance concentrations in short-term experiments, as opposed to only one test concentration used by Tomlinson et al. (1966). Therefore, Wood et al. (1981) were able to identify “no-effect concentrations” besides inhibitory test substance concentrations. Thus the NOEC of 0.38 mg/L is carried forward to the risk assessment.