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Toxicity to microorganisms

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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.