3',5'-dichloro-4'-ethyl-2'-hydroxypalmitanilide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1994, december 13th to 1995, january 11th

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to the Test Guidelines described in the EEC Directive 84/449/EEC.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3',5'-dichloro-4'-ethyl-2'-hydroxypalmitanilide

EC Number:

406-200-8

EC Name:

3',5'-dichloro-4'-ethyl-2'-hydroxypalmitanilide

Cas Number:

117827-06-2

Molecular formula:

C24H39Cl2NO2

IUPAC Name:

N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)hexadecanamide

Details on test material:

A bulk sample (stated weight 300 g) of the test material was
supplied by the Study Sponsor, arriving at Pharmaco LSR 26 July
1994. The test material was cream in colour and granular in form
and was identified as M-3221, batch number 944002. The sample was
stored at ambient temperature and protected from light until use
in testing.
The identity, strength and purity of the test material received
and its stability under the storage conditions were the
responsibility of the Sponsor.
Nominal exposure concentrations quoted in this report refer to the
test material as received; no allowance has been made for a purity
of less than 100%.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Axenic, uni-algal slope cultures of Selenastrum
capricornutum, CCAP number 278/4 were obtained from the
Culture Collection of Algae and Protozoa (CCAP), Freshwater
Biological Association, Ferry House, Ambleside, Cumbria,
2 November 1994. These slopes were stored in an
illuminated incubator nominally at 5°C until required.
Initially, liquid cultures for the test inoculum were
established by inoculation of OECD mineral salts medium
(50 ml; Appendix 1) with cells aseptically removed from
slope cultures. Subsequently, subcultures were prepared
from these liquid cultures by the aseptic transfer of
aliquots into fresh OECD medium, which was then incubated
for several days, until the required cell density had been
achieved.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

Ammonium chloride (NH4Cl) 15.0 mg/l
Magnesium chloride (MgCl2.6H20) 12.0 mg/l
Calcium chloride (CaCl2.2H20) 18.0 mg/l
Magnesium sulphate (MgSO4.7H20) 15.0 mg/l
Potassium dihydrogen orthophosphate (KH2PO4) 1.6 mg/l
Ferric chloride (FeCl3.6H20) 0.08 mg/l
EDTA disodium salt (Na2EDTA.2H20) 0.1 mg/l
Boric acid (H3B03) 0.185 mg/l
Manganous chloride (MnCl2.4H20) 0.415 mg/l
Zinc chloride (ZnCl2) 0.003 mg/l
Cobaltous chloride (C0Cl2.6H20) 0.0015 mg/l
Cupric chloride (CuCl2.2H20) 0.00001 mg/l
Sodium molybdate (Na2MoO4.2H20) 0.007 mg/l
Sodium hydrogen carbonate (NaHCO3) 50.0 mg/l

Test temperature:

The temperature of the incubator ranged from 21.2 to 24.8°C during
the test.
The temperatures of the contents of control and test flasks at the
start of the test ranged between 21.3 and 21.8°C; after 72 hours,
temperatures ranged from 22.4 to 22.8°C.

pH:

The pH of the contents of the control and test flasks measured at
the start of the test ranged from 7.4 to 7.6 respectively; after
72 hours pH ranged between 7.3 and 7.8.

Nominal and measured concentrations:

nominal concentration : 0.1, 0.2, 0.5, 1, 2, 5 and 10 mg/l
measured concentrations : 0 and 5,68 mg/l

Details on test conditions:

On the day of preparation, the test medium was clear and
colourless with particulate material visible on its surface and on
the bases of the vessels.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 5.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 5.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Cell counts in control and test cultures in the rangefinding test
indicated that the 72-hour EC5O of M-3221 to Selenastrum capricornutum
was greater than 100 mg/l (nominal value).
The results of chemical analysis indicated that M-3221 was poorly
soluble in the mineral salts medium; analysis of samples of filtered
media was not attempted because a method for extracting M-3221 from
water at low levels (< 1 mg/l) could not be identified. Based on
visual observations, the limit of aqueous solubility was less than
1 mg/l. Analysis of unfiltered samples of test media indicated that at
1 and 10 mg/l the levels achieved were adequately maintained; measured
concentrations at the other levels were variable.
4.1 Chemical analysis
The results indicate that the exposure concentration, although
lower than intended (52% of its nominal value), was adequately
maintained during the test; the overall mean measured level of
M-3221 was 5.68 mg/l.
4.2 Algal growth
Exposure of Selenast rum capricornutum to M-3221 at a mean measured
concentration of 5.68 mg/l did not result in a significant
reduction (p >0.05) in either the average specific growth rates or
biomass of test cultures compared to solvent control cultures.
Thus, the no-observed-effect concentration (NOEC) for growth rate
and biomass was not identified but must be >5.68 mg/l and the
median effect concentrations (ErC5O and EbC5O) could not be
calculated.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

valid study

Conclusions:

Cell counts in control and test cultures in the rangefinding test
indicated that the 72-hour EC5O of M-3221 to Selenastrum capricornutum
was greater than 100 mg/l (nominal value).
The results of chemical analysis indicated that M-3221 was poorly
soluble in the mineral salts medium; analysis of samples of filtered
media was not attempted because a method for extracting M-3221 from
water at low levels (< 1 mg/i) could not be identified. Based on
visual observations, the limit of aqueous solubility was less than
1 mg/i. Analysis of unfiltered samples of test media indicated that at
1 and 10 mg/i the levels achieved were adequately maintained; measured
concentrations at the other levels were variable.