Dichlone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study under consideration of GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Triplicate samples from the freshly prepared test media (containing
algae) of all test concentrations and from the control were taken at
the start of the test.
For the determination of the stability of the test item under the test
conditions and of the maintenance of the test item concentrations
during the test period, triplicate samples were taken after 24 and 48
hours test duration from each of the three replicates of all treatment
groups and at the end of the test (after the 72 hours test period) by
pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of 2 with acetonitrile.
One additional sample of the blank control and one sample of the
dilution solvent were taken at test start, 24, 48 hours and test end
without any sample treatment.

Storage of Samples:
All samples were stored (at April 07 respectively 08, 09 and 10, 2015)
in a freezer (≤ - 10 °C), protected from light until analysis was
performed (May 18, 2015).

Fortified Samples:
Approximately 50 mg of the test item were dissolved (1 minute
ultrasonication) in 50 mL acetonitrile to obtain a stock solution of
approximately 1 g test item/L. Four stock solutions were prepared.
Appropriate amounts of these stock solutions were diluted with test
water to obtain fortified samples at a level of 18, 25, 50 and 2800 μg
test item/L. Exact values were documented in the raw data.

Vehicle:

no

Details on test solutions:

The test item was not easily soluble in test water. In this study, only
the inhibitory effects of dissolved test item was tested and thus no
concentrations above the solubility limit of the test item in test water
were tested. Therefore, a stock suspension of 2000 μg test item/L
was prepared by suspending 2100 μg test item in 1050 mL test water
for preparing the test concentrations. The stock suspension was
treated with ultrasound for 10 minutes and stirred for 24 hours at
room temperature to dissolve as much test item as possible. Then,
non-dissolved fractions of the test item were separated from the test
medium by membrane filtration (0.45 μm cellulose nitrate filter). The
solution with dissolved test item was used as the test medium of the
highest test concentration and adequate volumes of this test medium
were diluted with test water to prepare the test media of the other
desired test concentrations.
The test media were prepared just before introduction of the algae (=
start of the test).

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
Origin: The algae were supplied by the Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
Reference Item: For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Water hardness of the test water: 0.621 mmol/L as CO3^2-

Test temperature:

22 to 23 °C

pH:

7.6 to 8.1 at test start and 8.1 to 9.4 at test end

Salinity:

n/a

Nominal and measured concentrations:

2000, 633, 200, 63, 20, 6.3 and 2 μg test item/L, corresponding to initial mean measured concentrations of 1740, 567, 202, 61.7, 18.8, 5.92 and 1.88 μg test item/L and time weighted mean measured concentrations of 911, 316, 102, 33.6, 10.0, 3.15 and 1.00 μg test item/L and a control.

Details on test conditions:

Water temperature: 22 to 23 °C; pH values at test start 7.6 to 8.1, at
the end of the test 8.1 to 9.4 ; continuous illumination; mean light
intensity: 5615 lux (5220 to 5950 lux).

The test item was not easily soluble in test water. In this study, only
the inhibitory effects of dissolved test item was tested and thus no
concentrations above the solubility limit of the test item in test water
were tested. Therefore, a stock suspension of 2000 μg test item/L
was prepared by suspending 2100 μg test item in 1050 mL test water
for preparing the test concentrations. The stock suspension was
treated with ultrasound for 10 minutes and stirred for 24 hours at
room temperature to dissolve as much test item as possible. Then,
non-dissolved fractions of the test item were separated from the test
medium by membrane filtration (0.45 μm cellulose nitrate filter). The
solution with dissolved test item was used as the test medium of the
highest test concentration and adequate volumes of this test medium
were diluted with test water to prepare the test media of the other
desired test concentrations.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

203 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. interval: 198-207

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

91.6 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. interval: 85.5-97.3

Details on results:

The microscopic examination of the shape of the algal cells after
72 hours of test duration did not show any difference between the
algae that had been growing up to a nominal test concentration of
20 μg test item/L and the algal cells in the control. Thus, the shape of
the algal cells was not obviously affected up to this test
concentration. The algal cells of the nominal concentrations of 63,
200, 633 and 2000 μg/L showed deformations compared to the algal
cells in the control

At the start of the test recoveries of the nominal test concentration
varied between 87 and 106% (of the nominal concentrations of 63,
200, 633 and 2000 μg test item/L). The nominal test concentrations
of 2, 6.33 and 20 μg test item/L could not be quantified as they were
lower than the limit of quantification (LOQ: 25 μg/L). After 72 hours
test duration, the recoveries of the nominal values varied between
18 and 25% (of the nominal concentrations of 633 and 2000 μg test
item/L). The test concentrations of 2, 6.33, 20, 63 and 200 μg test
item/L could not be quantified as these concentrations were below
the limit of quantification.

Results with reference substance (positive control):

72h EC50 = 0.845 mg/L (growth rate)

Validity criteria fulfilled:

yes

Description of key information

Very toxic (acute effects) for algae. 

Additional information

The toxicity to aquatic algae and cyanobacteria was assessed in a 72h study conducted according to OECD guideline 201 and under consideration of GLP. The nominal test concentrations were 2000, 633, 200, 63, 20, 6.3 and 2.0 μg test item/L. The test concentrations were confirmed by specific analysis. The quantification of the test item was performed using liquid chromatography with UV detection. At the start of the test recoveries of the nominal test concentration varied between 87 and 106% (of the nominal concentrations of 63, 200, 633 and 2000 μg test item/L). The nominal test concentrations of 2, 6.33 and 20 μg test item/L could not be quantified as they were lower than the limit of quantification (LOQ: 25 μg/L). After 72 hours test duration, the recoveries of the nominal values varied between 18 and 25% (of the nominal concentrations of 633 and 2000 μg test item/L). The test concentrations of 2, 6.33, 20, 63 and 200 μg test item/L could not be quantified as these concentrations were below the limit of quantification. All reported values refer to initial mean measured concentration. According to the OECD test guideline, the alga growth inhibition test is a more dynamic test system than most other short-term aquatic toxicity tests. As a consequence, the actual exposure concentrations may be difficult to define, especially for adsorbing substances tested at low concentrations. The estimated logKoc of the compound is 3.1 and 3.2, respectively (EPISuite MCI and logKow method). The compund is therefore expected to adsorb. In such a case, disappearance of the test substance from solution by adsorption to the increasing algal biomass does not mean that it is lost from the test system. Therefore, it is appropriate to base the analysis of the results on the initial measured concentrations.

The ErC50 was determined to be 203 μg/L and the ErC10 was determined to be 91.6 μg/L.

Additionally, several publications are available for the compound's toxicity towards algae. A study from the MInistry of the Environment, Japan revealed an EC50 of 0.072 mg/L. Pal et al. investigated the effect of the compound on Spyrogyna sp. At a concentration of 0.88 ppm all algae died after an observation time of 24h. Furthermore, the inhibiting effect on algal growth was shown in a study published by Nilson et al. A concentration of 1 mg/L resulted in approx. 100% inhibition of P. inundatum. The growth rate of a second species (C. pyrenoidosa) was inhibited by >90% at concentrations of 25.0 and 5.0 mg/L. At a concentration of 1.0 mg/L around 40% inhibition could observed.

In conclusion, the compound is very toxic for algae.