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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data based on the inhibition control of a ready biodegradability study. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2012).
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
17 Jul 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany. 08 Apr 2015.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Composition of medium: Mineral medium prepared from stock solutions a (KH2PO4), b (CaCl2), c (MgSO4*7H2O), and d (FeCl3*6H2O)
- Test temperature: 19.5 - 21.6 °C
- pH: 6.7 - 7.3 (pH at the end of the test, before addition of HCl)
- Suspended solids concentration: 25.0 mg dry matter/L
- Continuous darkness: Yes.
Test organisms (species):
other: activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, NW-Lachen-Speyerdorf, Germany. The plant treats mostly domestic sewage. Date of collection: 28 Oct 2016.
- Pretreatment: The sludge was filtered, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use.
- Concentration of sludge: The dry matter was determined as 3700 mg suspended solids/L.
- Initial inoculum concentration: 25.0 mg/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
19.5 - 21.6 °C
pH:
6.7 - 7.1
Details on test conditions:
TEST CONDITIONS
- Composition of medium: Mineral medium prepared from stock solutions a (KH2PO4), b (CaCl2), c (MgSO4*7H2O), and d (FeCl3*6H2O)
- Test temperature: 19.5 - 21.6 °C
- pH: 6.7 - 7.3 (pH at the end of the test, before addition of HCl)
- Suspended solids concentration: 25.0 mg dry matter/L
- Continuous darkness: Yes.

TEST SYSTEM
- Culturing apparatus: Schott-flasks and 100 mL scrubber flasks as CO2 absorbend vessels.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The test vessels were aerated for 72 h with purified (by activated charcoal), CO2-free (CO2-scrubbed), moistened air to purge the sysem of CO2.
- Measuring equipment: Carbon analyser TOC multi N/C 2100S, Analytik Jena
- Test performed in open system: Yes.
- Details of trap for CO2 and volatile organics if used: The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask.
- Other: The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.

SAMPLING
- Sampling frequency: Samples were taken on Day 0, 2, 4, 7, 9, 11, 14, 18, 23, and 29.
- Sampling method: 1 mL was collected. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2.
- Other: On Day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2. On Day 29, samples from both scrubber flasks were taken.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2, containing mineral medium and inoculum
- Apparatus blanks: 2, containing mineral medium only
- Abiotic sterile control: 1, containing test item, mineral medium and HgCl2
- Toxicity control: 1, containing test item, positive control, mineral medium and inoculum.
- Positive control: 2, containing positive control, mineral medium and inoculum
Reference substance (positive control):
yes
Remarks:
aniline
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
25.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Result of toxicity control from ready biodegradability test
Results with reference substance (positive control):
Degradation of the positive control was 70 % after 9 days.

The toxicity control attained 45.1% degradation after 14 days of incubation. According to the guideline a substance can be considered as not inhibitory if more tahn 35% (based on total DOC) occured within 14 days.

Since the substance was degraded by 45.1% after 14 days it is with high probability not toxic to aquatic microorganisms. The test item concentration in the toxicity control of 25.5mg/L can be used as NOEC value for the toxicity to aquatic microorganisms.

Table 1: Degradation toxicity control

Day

Toxicity Control [%]

2

0.7

4

21.2

7

35.6

9

38.7

11

43.0

14

44.1

18

44.6

23

45.5

29

45.7

 

Description of key information

Not toxic to aquatic microorganisms

Key value for chemical safety assessment

Additional information

Studies on the toxicity of 2,5-bis(1,1,3,3-tetramethylbutyl)benzene-1,4-diol to aquatic microorganisms are not available. Therefore the assessment is based on the results of an inhibition control of the available ready biodegradability study according to OECD 301B. The test substance concentration in the inhibition control is used to derive a NOEC for the toxicity to aquatic microorganisms. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2017). A substance can be assumed to be not inhibitory to aquatic microorganisms, if in the inhibition control of a ready biodegradation test, more than 25% degradation based on [oxygen demand (BOD/ThOD) or CO2 evolution (CO2/ThCO2)] occurred within 14 days (OECD guideline 301). The inhibition control of the available test contained 25.5 mg/L test substance and 26.2 mg/l reference substance (aniline). The degradation rate was 45.1% after 14 days. The test substance concentration of 25.5 mg/L is taken as NOEC (14 d).

Based on the results the substance is considered to be not toxic to microorganisms.