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Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Days
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
July 1992
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
diethyl({3-[(4-nitrophenyl)formamido]propyl})azanium chloride
EC Number:
Cas Number:
Molecular formula:
diethyl({3-[(4-nitrophenyl)formamido]propyl})azanium chloride
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Arrival: 7 November 2017
Internal code: 17/5739
Substance name: N-[3-(Diethylamino)propyl]-4-nitrobenzamide, hydrochloride
Synonym: “Nitrobenzamid x HCl”
Chemical name: -
EAS material container code: MC-00013846-17-0001
EAS Study code: S17-08075
CAS number: 110393-28-7
EINECS: Not available
Molecular formula: C14H22N3O3Cl
ThOD (NH4) 1.57 mgO2/mg PG (calculated on the base of molecular formula)
ThOD (NO3) 2.18 mgO2/mg PG (calculated on the base of molecular formula)
Molecular weight: 315.8 g/mol
Purity of main component: 94.2% w/w
Batch/Lot number: Op 1/16 and Op 2/16
Stability: stable
Water solubility: soluble into water.
Volatility: No data available
Density: No data available
pH: 3 - 5
Appearance: Powder
Colour: offwhite
Storage conditions: Room temperature
Expiration date: 18 April 2019
Certificate of analysis: Available from 18 April 2017
Origin: Clariant Plastics & Coatings (Deutschland) GmbH
Identification: Colour: offwhite; consistency: powder

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
Activated sludge from the municipal wastewater treatment plant Staufener Bucht was used as test system. The treatment plant has a capacity of 140 000 inhabitant equivalents and clarifies predominantly domestic wastewater. Sampling date of activated sludge was 21 February 2018. The activated sludge was kept aerated until use at test temperature. The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in chlorine-free tap water. Dry solid of the activated sludge was determined as 4.8 g/L by weight measurements before and after drying at 105°C (mean of triplicate measurements).
Duration of test (contact time):
ca. ca. 28 d
Initial test substance concentration
Initial conc.:
45.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The system OxiTop®- Control from WTW, Weilheim and the Sensomat-System from Aqualytic GmbH & Co., Langen were used as test systems. They consists of narrow-necked glass bottles with rubber sleeve inserts for NaOH pellets, in which the carbon dioxide evolved is absorbed. The liquid volume was fixed as 164 mL each. Magnetic stir bars were introduced into the bottles which were positioned on a stirrer platform. The bottles were sealed tightly with the measuring heads with a measuring range of 500 – 1350 hPa. Every 112 minutes the current pressure was measured and stored by each measuring head. At the end of the experiment pressure data were read out via an infrared interface to the controller unit and afterwards data were transferred via a RS232 interface using the Achat OC software of WTW to an Excel file where further data processing was carried out. Additionally several measured values were randomly read out and recorded by hand and were afterwards compared with the printed excel table for quality control.
In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound (reference) and one reactor containing reference compound and test item (toxicity control) were set up.
The test flasks were filled as described in paragraphs 5.4 – 5.7 and tempered to the incubation temperature for one hour. Afterwards, 1025 µL of the inoculum were added into each flask, one sodium hydroxide pellet was added to each rubber quiver inserted in the flasks and the flasks were sealed tightly with the measuring heads and the test was started. After 28 days the data were read out and the pH in the flasks was measured
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Key result
% degradation (O2 consumption)
ca. 39.3
Sampling time:
28 d
Details on results:
The standards evaluation of the OECD 301 F assumes that nitrification of the nitrogen content takes place unless, it can be proven, by additional nitrogen analysis, that the nitrogen was not oxidised. The reference point for biodegradability is therefore ThOD(NO3).
The biodegradation of the test item was 32.9% within 28 days, based on the assumption, that the test item nitrogen was metabolized to NO3 (mean of three replicates, see table 2 and figure 1).
Degradation based on ThOD (NH4)
Due to the long lag phase, which could also influence nitrification an informative additional evaluation assuming that no nitrification took place has been carried out.
The biodegradation of the test was 45.7% within 28 days, based on the assumption, that the test item nitrogen was metabolized to NH4 (mean of three replicates, see table 4 and figure 2).
For both evaluations the test item didn’t reach the criteria for ready biodegradability (60% of ThOD within a 10-d window).
The pH in the test vessels at the end of the test was 7.4 – 7.5.
Toxicity control
For both evaluations (ThOD(NO3) and ThOD(NH4)) the degradation extent in the toxicity control reached a value higher than 25% within 4 days (see table 2 and 4). According to the validity criterion of the guideline OECD 301 the test item had no toxic effect to the inoculum.
The pH in the toxicity control vessel at the end of the test was 8.6.
The oxygen consumption of the blanks was 29.0 mg/L in 28 days (mean of three replicates, see table 1).
The pH in the blank vessels at the end of the test was 7.6.

BOD5 / COD results

Results with reference substance:
Reference item
The reference compound sodium acetate reached the pass level for ready biodegradability (60% ThOD within a 10-day window) within 4 days (75.0%, see table 2).
The pH in the reference vessels at the end was 9.1 – 9.2.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable