Ichthammol

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of study: March 10th, 1995
Termination of study: April 7th, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

The study was carried out according to the EC guideline L 383 A: C.4-E.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Ichthammol

EC Number:

232-439-0

EC Name:

Ichthammol

Cas Number:

8029-68-3

Molecular formula:

unspecified

IUPAC Name:

Ichthammol (UVCB substance)

Test material form:

liquid: viscous

Details on test material:

sample from a batch from regular production released for use as an active pharmaceutical ingredient in medicinal products.

Specific details on test material used for the study:

Batch no. R 954623 from regular production at the registrant's manufacturing site.

Ichthammol is characterized in the study report with trademark ICHTHYOL.

Release of substance was based on testing according to pharmacopoeia specifications/instructions of ÖAB / PH.EUR or DAB 10. A certicate of analysis is attached to the original study report.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Remarks:

pre-conditioned to the experimental conditions

Details on inoculum:

Secondary effluent from a treatment plant
Inoculation was made using a secondary effluent of good quality collected from a treatment plant dealing with predominantly domestic sewage. The effluent was kept under aerobic conditions in the period between sampling and application. To perpare the inoculum the sample was filtered through a coarse filter, the first 200 ml being discarded. The rest of the filtrate was kept aerobic until use. The inoculum was used on the day of collection. The number of bacteria was determined with Easicult TTC. There were approximatley 10^5 bacteria per litre of final volume of the test medium.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

2 mg/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Principle
A predetermined amount of the compound is dissolved in an inorganic medium (mineral nutrient medium), providing an usual concentration of 2 mg active substance per litre (AS/l). The solution is inoculated with a small number of micro-organisms from a mixed population and kept in closed bottles in the dark in a constant temperature bath at 20°C ± 1°C. The degradation is followed by oxygen analyses over a 28-day period. A control with inoculum, but without test material, is run parallel for the determination of oxygen blanks.

Reagents
Dilution water:
Distilled and desalinated water - containing not more than 0.01 mg Cu/I.

Nutrient medium:
Solution 1: 0.850 g KH2PO4; 2.850 g K2HPO4 -3H2O; 3.340 g Na2HPO4 -2H2O; 0.050 g NH4Cl
were dissolved in distilled water and made up to 100 ml.
Solution 2: 2.25 g MgSO4 • 7H2O were dissolved in distilled water and made up to 100 ml.
Solution 3: 3.64 g CaCl2 • 2H2O were dissolved in distilled water and made up to 100 ml.
Solution 4: 0.025 g FeCl3 • 6H2O were dissolved in distilled water and made up to 100 ml.

25 ml each of solutions 1 to 4 was added to aerated distilled water to give a total volume of 25 l mineral nutrient medium. The pH was 7.46. 24 hours before use the mineral nutrient medium was vigourously air saturated, at room temperature (19.0°C), for 20 minutes with compressed air. After standing for 24 hours the oxygen concentration at 19.8°C was 8.9 mg O2/I. All transfer and filling operations of the air saturated water were conducted bubble-free by siphon.

Inoculation
The following inoculum was used: Secondary effluent from a treatment plant.
Inoculation was made using a secondary effluent of good quality collected from a treatment plant dealing with predominantly domestic sewage. The effluent was kept under aerobic conditions in the period between sampling and application. To prepare the inoculum the sample was filtered through a coarse filter, the first 200 ml being discarded. The rest of the filtrate was kept aerobic until use. The inoculum was used on the day of collection. The number of bacteria was determined with Easicult TTC. There were approximately 10^5 bacteria per litre of final volume of the test medium.

Procedure
Test system
Substance: Ichthyol (trademark name for Ichthammol)
Concentration: 2 mg/l
Control substance: sodium acetate
Concentration: 4 mg/l
Vehicle for the test and control substance: test medium
Oxygen measuring instrument: OXI DIGI 530-3 with a Tri Oxmatic - EO 200 oxygen electrode
Duration of test: 28 days

Parallel groups of BOD bottles (300 ml) were prepared for the determination of the test and reference chemicals in simultaneous experimental series, including inoculum blanks. The following times were used: 0, 3, 7,10,14,17, 21, 24 and 28 days.
The test was conducted in duplicate in a parallel series.
Fully aerated mineral nutrient medium was added to large bottles so that they were about one-third full. Then sufficient amounts of the stock solutions of the test chemical and reference chemical were added to separate large bottles so that the final concentration of the chemicals was 2 mg/litre. No chemicals were added to the blank control medium contained in a further large bottle.
The solutions were inoculated from a pointed pipette with 2000 µl/l secondary effluent per litre test medium. The solutions were made up to volume with aerated mineral nutrient medium using a hose which reaches down to the bottom of the bottle to achieve adequate mixing.

Subsequently each prepared solution was dispensed into the respective group of BOD bottles (300 ml) by hose from the lower quarter (not the bottom) of the appropriate large bottle so that all the BOD bottles were completely filled.
The bottles were gently tapped to remove any air bubbles. Two bottles were immediately analysed for dissolved oxygen. The remaining replicate bottles were stoppered ensuring that no air bubbles were enclosed and placed in a water bath at 20.0°C - 20.1°C, kept in the dark and removed in duplicate after 3, 7, 10, 14, 17, 21, 24 and 28 days for immediate dissolved oxygen analysis.

Inhibition test:
No inhibition test was carried out, as the acute toxicity test for Pseudomonas putida revealed a low bacteriotoxicity of EC50 (16 h) > 1000 mg Ichthyol/l (see LPT Report No. 9151/95). The concentration employed in this test was 2 mg/l.

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

Preliminary study:

no preliminary study (see "details on study design")

Test performance:

No unusual observations or occurences affecting results (see "details on study design")

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

7.9

Sampling time:

28 d

Details on results:

The purpose of this test is the measurement of the biodegradability of Ichthyol^ in an aerobic, aqueous medium at a concentration of 2 mg test substance/l.

Test system
Analysis (Oxygen electrode): OXI DIGI 530 with a Tri Oxmatic - EO 200 oxygen electrode
COD of test material: 1.26 mg 02/mg test material
ThOD of sodium acetate: 0.78 mg 02/mg sodium acetate
Temperature of the dilution water after aeration: 19.8°C
O2-concentration of the water (after aeration and standing before start of test): 8.9 mg O2/l
Temperature of water bath: 8.9 mg O2/I 20.0°C - 20.1°C

BOD = biochemical oxygen demand
COD = chemical oxygen demand
ThOD = theoretical oxygen demand

BOD = (mt0-mtx)-(mb0-mbx)/mgsubs/l

mb0 = mean of dissolved oxygen in mg O2/I after 0 days (blank)
mbx = mean of dissolved oxygen in mg O2/I after x days (blank)
mt0 = dissolved oxygen in mg O2/I after 0 days (test substance)
mtx = dissolved oxygen in mg O2/I after x days (test substance)
mgsubs = mg test substance used

%degradation = BOD/COD or ThOD x 100

02-determination
Mineral nutrient solution without test material and with inoculum (mean blank):
8.84 mg O2/l after 0 days (mb0) and 7.83 mg O2/l after 28 days (mb28)
Mineral nutrient solution with sodium acetate and with inoculum (mean control):
8.84 mg O2/l after 0 days and 5.26 mg O2/l after 28 days
Mineral nutrient solution with Ichthyol and with inoculum (mean test material):
8.84 mg O2/l after 0 days and 5.26 mg O2/l after 28 days
8.82 O2/l after 0 days (mt0) and 7.62 mg O2/l after 28 days (mt28)
(further "interim" results after x days in original study report")

Dissolved oxygen (DO) depletion: % degradation
control chemical sodium acetate (mean): 82.5% after 28 days
test material Ichthyol (mean): 7.9% after 28 days
(further "interim" results after x days in original study report)

Validation of results
As required by the guidelines the dissolved oxygen depletion (DO) of the blank (mb0 to mb28) did not exceed 1.5 mg dissolved oxygen/litre after 28 days.

Test substance
Control chemical: sodium acetate
Result: % degradation after 28 days: 82.5%
Test material: Ichthyol
Result: % degradation after 28 days: 7.9%

Under the present test conditions, only a marginal biodegradability was determined for Ichthyol within 28 days.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The purpose of this test was the measurement of the biodegradability of Ichthyol in an aerobic, aqueous medium at a concentration of 2 mg test substance/l. Under the present test conditions, only a marginal biodegradability was determined for Ichthyol (trademark name of Ichthammol) within 28 days.