Semicarbazide hydrochloride

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

october 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

In the range-finding test, the 2500 mg/L test item stock solution was cooled to 24 ºC not 20 ± 2ºC as stated in the Study Plan.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Semicarbazide hydrochloride
Batch: 55507861
Purity: 99.3%
Physical state/Appearance: white solid
Expiry Date: 28 April 2019
Storage Conditions: room temperature in the dark

Analytical monitoring:

not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dissolved directly in water.
A nominal amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity.

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 100, 180, 320, 560 and 1000 mg/L.
The test item was dissolved directly in water.
An amount of test item (5000 mg) was dissolved in water and the volume with the aid of ultrasonication for approximately 5 minutes adjusted to 2 liters to give a 2500 mg/L stock solution. Test concentrations were prepared by taking aliquots ((20, 36, 64, 112 and 200 mL (3 replicates of each)) of the 2500 mg/L stock solution and dispersing with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentrations of 100, 180, 320, 560 and 1000 mg/L (3 replicates of each).

- Eluate: The test water used for the range-finding and definitive tests was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 27 September 2017 for the range-finding test and on 20 December 2017 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
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 a final volume of 1 liter of water with the aid of ultrasonication.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not specified

Test temperature:

20-22°C

pH:

7.8

Dissolved oxygen:

initial O2: between 2.3 and 7.7 ,g O2/L

Salinity:

none

Conductivity:

not specified

Nominal and measured concentrations:

nominal concentrations: 0, 10, 100, 1000 mg/L (range-finding test)
nominal concentrations: 0, 100, 180, 320, 560, 1000 mg/L (final test)

Details on test conditions:

TEST SYSTEM
Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (9.5 liters) was fed synthetic sewage (475 mL). The pH of the sample on the day of the test was 7.6 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. Two additional control vessels were then prepared prior to the test item vessels as described in Section 3.4.2.1. Finally, two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 20 °C and 22 °C.

Measurement of the Respiration Rates
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).
- Particulate matter: The suspended solids concentration was equal to 3.0 g/L prior to use.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: normal laboratory lighting
- Light intensity: normal laboratory lighting

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 120 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. 760 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 8.5 mg/L.
The specific respiration rate of the controls was 29.22 mg oxygen per gram dry weight of sludge per hour.
The coefficient of variation of oxygen uptake in the control vessels was 6.19 %.
All validation criteria for the study were therefore satisfied.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels:
EC10 = 1.8 mg/L
EC50 = 8.5 mg/L

Reported statistics and error estimates:

No statistically significant toxic effects were shown at the test concentration of 100 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentrations of 180, 320, 560 and 1000 mg/L. Based on these results the NOEC after 3 hours exposure was 100 mg/L.

None

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 760 mg/L, 95% confidence limits 580 to 1000 mg/L. The NOEC after 3 hours exposure was 100 mg/L.
The reference item gave a 3-Hour EC50 value of 8.5 mg/L, 95% confidence limits 6.5 to 11 mg/L.

Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 100, 180, 320, 560 and 1000 mg/L (3 replicates) for a period of 3 hours at a measured temperature of between 20 °C and 22 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-Dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 760 mg/L, 95% confidence limits 630 to 920 mg/L*. The NOEC after 3 hours exposure was 100 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

760 mg/L

EC10 or NOEC for microorganisms:

120 mg/L