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Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
8 July to 5 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 219 (Sediment-Water Chironomid Toxicity Test Using Spiked Water)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were made on additional replicate systems at day 0 and day 28 for determination of test substance concentrations in overlying water.
Vehicle:
no
Details on sediment and application:
Fine industrial sand, washed, was used as an inorganic substrate for the larvae

Test chambers were prepared 1 day before introduction of organisms, maintained under test conditions
Test organisms (species):
Chironomus riparius
Details on test organisms:
Laboratory cultures used to provide 1st instar (1-day old) individuals at test initiation. Age was confirmed by measurement of head capsule widths of a representative sample.

Breeding conditions: Cultured at room temperature (21-25 degrees C) in laboratory culture water.
Handling of egg masses and larvae: Egg masses collected and isolated in freshwater, maintained at 20 degrees C.
- Feeding during test: prepared invertebrate food- a suspension of commercial fish flake food
- Amount: Daily, 2.5-3 ml of 2.0 mg/ml during 1st 10 days, 6-8 ml for remainder
Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
no
Duration:
28 d
Exposure phase:
total exposure duration
Hardness:
140 to 162 mg CaCO3/L
Test temperature:
19.4 to 21.7 degrees C
pH:
7.5 to 8.7
Dissolved oxygen:
5.2 to 9.3 mg/L (60 to 95% saturation)
Ammonia:
0.00003 to 0.0168 micrograms/L (un-ionized)
Nominal and measured concentrations:
Nominal concentrations definitive test : 0 (control), 2.5, 5.0, 10, 20, 40 and 80 mg B/L.
Geometric Mean Measured Overlying Water Total Concentrations: 0.298, 3.06, 5.93, 11.0, 20.4, 43.3, and 89.3 mg B/L
Geometric Mean Measured Overlying Water Added Concentrations: 0, 2.76, 5.63, 10.7, 20.1, 43.0, and 89.0 mg B/L
Details on test conditions:
Test chambers were 1-L glass jars containing approximately 255 g of sediment (clean washed sand) Sediment depth was approximately 2 cm. Dilution water (600 mL) was added to each replicate chamber using a deflector to minimize disturbance of the sediment. Water depth was approx 8 cm, so the water: sediment volume ratio was approx 4:1. Polypropylene emergence traps with nylon mesh were used to monitor adult midge emergence. Chambers were left for 1 d to equilibrate before the test started.

At least 7 replicate chambers were prepared for each treatment level and control. Two were used to provide samples for chemical analysis and 3 chambers were used for biological observations. 20 midge larvae were added to each chamber for the chemical and biological observations. Seven different exposures were tested, a control and six treatments. Larvae were impartially added to cups, then the cups randomly assigned to a replicate chamber.

Overlying water was aerated during the test beginning 24 h after addition of the organisms by gentle bubbling. Midges were fed 2.5 to 3 ml of a commercial flake fish food suspension (2.0 mg/ml) for the first 10 d, then 8 ml daily for the remainder of the test. Algae were added during the first 3 days as food for the first instar larvae. Lighting was 16 h: 8 h light:dark cycle with a 30 m transition period. Light intensity was 520 lux. Temperature was maintained at 20 +/- 2 degrees C by keeping the test chambers in a flowing water bath. Water was not replaced during the test.

Emergence of adult flies was monitored from day 18. Presence of adults or exuviae in traps was monitored. Adult flies were identified by gender.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
20.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
20.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: emergence
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: emergence
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
development rate
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
emergence rate
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
40.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: total adult emergence
Remarks on result:
other: added boron (30.2 - 50.8)
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
42.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: mortality and emergency
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: mortality, emergence and development rate
Remarks on result:
other: added boron
Details on results:
The nominal concentrations were added in the effect concentrations table, following the usual practice that a nominal concentration may be used if the measured values are reasonably close to the nominal.

Percent emergence in the controls was 97% which exceeded the minimum of 70% emergence specified in the OECD 219 guideline for valid results.

All chemical and physical parameters for the 28 day study were within expected ranges, except for a 9 h period where temperature rose to 22.6 degrees due to water bath heater malfunction. This was judged to have no effect on the results as control performance met criteria and all treatments were equally affected.

Light intensity: 520 lux (day 0).
Reported statistics and error estimates:
Statistical analysis based on one-way ANOVA followed by Dunnett's test. ECx estimates obtained from probit method and trimmed Spearman-Karber method if poor fit in the probit approach.

Measured Overlying Water Concentrations and Endpoint Values

Group Nominal Exposure (mg-B/L) Measured Day 0 Exposure (mg-B/L) Measured Geomean Exposure (mg-B/L)

 Mean measured added boron

Survival  Emergence  Gender Ratio*  Development rate    
Control  0 0.429 0.298

 0

98% 97% 1.6 0.048
T1  2.5 3.22 3.06 2.76 98% 95% 0.5 0.044
T2  5 6.03 5.93 5.63  100% 98% 0.5 0.045
T3  10 10.4 11 10.7 97% 92% 0.8 0.045
T4  20 21.6 20.4 20.1  100 90% 0.5 0.043
T5  40 44.3 43.3 43.0  87%** 80%** 0.9 0.044
T6  80 87.6 89.3 89.0  0%** 0%** -- --**

* Ratio is males:females

* Significantly different from control value (p<=0.05)

The gender ratio was reduced in all treatments above the control. However, there was no dose-related pattern; the most highly exposed group (T5) with emergent flies was most similar to controls. In addition, the test with spiked sediments showed virtually the reversed pattern of gender ratio, again with no evident dose-response pattern.

Validity criteria fulfilled:
yes
Conclusions:
Expressed as nominal added boron the NOEC values for survival and percent emergence were 20 mg B-added/L and LOECs were 40 mg B-added/L. The NOEC value for development rate was 40 mg B-added/L.

Based on survival and percent emergence, the NOEC (total) was 20.4 mg B/L and LOEC (total) was 43.3 mg B/L. The EC10 (total) for survival was 43.0 mg B/L. The EC10 (total) for percent emergence was 40.8 mg B/L (95% CI 30.5 to 51.1 mg B/L) The NOECs (total) for time to emergence for males and females (calculated separately and combined) were 43.3 mg B/L. The NOECs (total) for development rate for males and females (calculated separately and combined) were 43.3 mg B/L.

Expressed as added boron the NOEC values for survival and percent emergence were 20.1 mg B-added/L and LOECs were 43.0 mg B-added/L. The NOEC value for development rate was 43.0 mg B-added/L. The EC10 for survival would be 42.7 mg B-added/L and the EC10 for percent emergence would be 40.5 mg B-added/L.

Analyses of the overlying water at test initiation and termination documented that the boron concentrations were relatively constant in the overlying water. This suggests that the silica sand sediment did not remove any significant amount of the boric acid spiked into the water.
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: test was carried out in conformity with the draft OECD document , May 1998 "Chironomid testing using spiked sediment" and OECD principles of GLP
Qualifier:
according to guideline
Guideline:
other: OECD draft document, may 1998
Deviations:
yes
Remarks:
the deviations are assumed not to have effected the results of the study.
Principles of method if other than guideline:
egg masses were not obtained from an own TNO culture.
The peat used was autoclaved before preparing the artificial sediment to better enable wetting.
extra nitrite and ammonia determinations were carried out a t=0.14 and 28days to confirm that these parameters did not disturb the results of the tests.
conditioning of the sediment, after mixing of the constituents, lasted 8d and not 7d as stated in Annex A of the protocol
an amendment to the protocol with respect tot the boron analyses was not prepared
according to the guideline the TOC of the artificial sediment should be 2 +/- 0.5% and not 2%
the TOC value in the artificial sediment was 2.73% instead of 2 +/- 0.5%
the pH KCl value of the artificial sediment (being 7.8) was above the intended value of 7.0 +/- 0.5
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- quantity: 1000 gram
- received from sponsor
- date of receipt in delft: 18 june 1999
- packing: one litre square polyethylene bottle
- Sample storage conditions before analysis: stored at room temperature, no protection from light
Vehicle:
no
Test organisms (species):
Chironomus riparius
Details on test organisms:
TEST ORGANISM
- Common name: non biting midge
- Source: egg masses were obtained form RIZA in lelystad, the netherlands
- Date of collection: Feb, 16 2000
- the hatched first instar larvae (larvae without hemoglobin) were used for the test



ACCLIMATION
- Acclimation period: Feb 16 --> Feb 19
- Equilibration period = 2 days
- Acclimation conditions : eggs were stored in a refrigerator, at that time they were placed in DSWL-E water for further development.
- Test started on Feb 25, 2000
- Feeding: larvae were fed daily with a dried fish food suspension (Aquariaan, 20g suspended in 100ml ultrapure water).
amount: 100 µl per test vessel, except at t=3,4,5,6,7,8 and 9 days (50 µl was given). The animals were not fed on Sundays, the double amount of food was given on Saturdays.
- The test vessels were covered with pieces of cloth from the 12th days to prevent escaping of emerged midges.

Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
no
Duration:
28 d
Exposure phase:
total exposure duration
Remarks:
=time needed for emergence of the adults in the control
Test temperature:
20.2 - 20.5 °C
pH:
artificial sediment: 7.8
overlying water: 7.1-8.1
Dissolved oxygen:
> 5.9 mg/l
Nominal and measured concentrations:
- Test substance concentrations: 18, 32, 56, 100, 180 and 320 mg B/kg dry weight of sediment d.w. 4 replicates were used.
- Control group: 32, 100 and 320mg/kg exposure concentrations
Details on test conditions:
TEST SYSTEM
- Test container (material, size): vessel=600mL beakers with diameter ca. 8cm
- Sediment volume: 1.5cm spiked sediment or control sediment/replicate container
- No. of organisms per container (treatment): 5 larvae per test vessel
- No. of replicates per substance concentration: 20 instar larvae
- Test vessels were covered by watch glasses during the test
- Equilibration period: 2 days


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (artificial substrate):
* 5-10% sphagnum peat, finely ground, no visible plant remains (66.4% of dry weight)
* 20% kaolin clay (99% of dry weight)
* 70-75% fine sand (100% of dry weight)
- Dry weight of artificial sediment: 59.6%
- The sediment/water systems were not replaced during the test

PHYSICO-CHEMICAL CHARACTERISTICS:
- artificial sediment:
pH-KCl = 7.8
TOC content = 2.73g C/100g dry weight
- overlying water:
pH = 7.1-8.1
lowest measured oxygen concentration: 5.9 mg/l
temperatur: 20.2-20.5°C
ammonia and nitrite concentrations were below the critical value for Chironomus riparius


PREPARATION
- Pretreatment of artificial and spiked sediment and of sediment/water exposure systems: prepared from an artificial sediment and the TNO standard water DSWL-E.
- Artificial sediment:dry weight 59.6%
- 4 replicate batches were prepared by mixing portions of 600f of clay, 2175g of sand and 225g of sphagnum peat. The peat was wetted the day before by addition of 1.5L of DSWL-E, followed by autoclaving for 20 minutes at 121°C. The clay and sand part were added together wit 0.5L of DSWL-E and 19g of CaCO3. The mixtures in the jars were homogenised by rolling them for 1 days on a roller bank, the content of the jars were added together, 1L of DSWL-E was added followed by aeration. The sediment was mixed and the dry weight of a sample was determined several times.
- After 8 days the sediment was mixed to obtain a homogenous distribution, before being placed in the exposure vessels and the overlying water was added.
- Sediments were spiked in the test beakers based on the dry sediment weight. For each exposure concentration a number of four replicate containers were separately filled with a layer of ca 1.5cm spiked sediment or controled sediment. For chemical analystical purposes 2 extra vessels were prepared for the control: 32, 100 and 320mg/kg exposure concentration. DSWL-E water was added to obtain a ratio between sediment layer and water column in the range of 1:4. this sediment/water system was allowed to settle for ca. 48h before the test animals were added. the overlying water of the sediment/water systems was slightly aerated during the test, without disturbing the sediment layer.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
180 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
180 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
emergence rate
Details on results:
OBSERVATIONS
- Some turbidity from suspended solids was observed during the test. De turbidity decreased during the test.
- From day 14 some solids were present on the walls of the test vessel; this pfenomenon increased during the remaining period of the test.


CHEMICAL ANALYSES:
- at the end of the test the boron concentrations in pore water and overlying water are similar; equilibrium between overlying water and pore water has been reached.
- at the start of the test the concentrations in pore water are higher than in the overlying water.

TOXICITY DATA
the 28d LC50 is 278 mg B/k dw (nominal), the 28d LOEC for mortality and emergence is 320 mg B/kg dw and the 28d NOEC for mortality and emergence is 180 mg B/kg dw.
Reported statistics and error estimates:
LC50 values and their interval were calculated by means of a parametric model (Kooiman, 1981).
Emergence data are arcsin transformed and the Dunnett test (sign level 99%) was used to detect significant differences with the control.

28d LOEC: 320 mg/kg (mortality & emergence)

28d LC50: 278 mg/kg

nominal conc. of mg B/kg sediment dw emerged larvae at the end of the testmale female total %0 34 42 76 95 18 34 44 78 98 32 36 37 73 91 56 35 43 78 98 100 34 44 78 98 180 42 37 79 99 320 1 1 2 2.5

Validity criteria fulfilled:
yes
Conclusions:
The test was carried out in conformity with the draft OECD document and OECD principles of GLP.
The test is well described.
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 June to 15 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were made on additional replicate systems at day 0 and day 28 for determination of test substance concentrations in overlying water, pore water and sediment.

Pore water was obtained by centrifugation of sediment at approx 4000 rpm for 20 min at 20 degrees C after removal of overlying water. Supernatant centrifuged at approx 9000 rpm for another 20 minute and analyzed for test substance.

Sediment concentration was measured after removal of pore water. Substance was extracted from sediment by mixing with reagent water then centrifugation and measurement of the supernatant.
Vehicle:
no
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Formulated sediment consisting of fine industrial sand (75% w/w), kaolin clay (20%) and fine sphagnum peat moss (5%)
- Method of mixing: Stock solutions prepared in water, aliquots added to each treatment batch of sediment and mixed by hand.
- Equilibration time: Test chambers prepared 2 days before introduction of organisms, maintained under test conditions
Test organisms (species):
Chironomus riparius
Details on test organisms:
Laboratory cultures used to provide 1st instar (1-day old) individuals at test initiation. Age was confirmed by measurement of head capsule widths of a representative sample.

Breeding conditions: Cultured at room temperature (21-25 degrees C) in laboratory culture water.
Handling of egg masses and larvae: Egg masses collected and isolated in freshwater, maintained at 20 degrees C.
- Feeding during test: prepared invertebrate food- a suspension of commercial fish flake food
- Amount: Daily, 2.5-3 ml of 2.0 mg/ml during 1st 10 days, 6-8 ml for remainder
Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
no
Duration:
28 d
Exposure phase:
total exposure duration
Hardness:
176 to 230 mg CaCO3/L
Test temperature:
18.8 to 20.9 degrees C
pH:
7.4 to 8.5
Dissolved oxygen:
4.7 to 9.0 mg/L (55 to 101% saturation)
Ammonia:
0.00003 to 0.0145 micrograms/L (un-ionized)
Nominal and measured concentrations:
Nominal Sediment Concentrations: 0 (control), 6.5, 13, 25, 50, 100, and 200 mg B/kg dry sediment
The day 0 measured concentrations of boron in the sediment samples were 0.207, 1.56, 4.27, 10.0, 18.1, 32.6, and 81.0 mg/kg dry sediment
The day 28 measured concentrations of boron in the sediment samples were
The initial (day 0) measured concentrations of boron within the overlying water samples were 0.319, 0.536, 0.818, 1.44, 2.96, 6.89, 14.4 mg B/L
The termination (day 28) overlying water measured concentrations were 0.125, 0.574, 1.42, 2.53, 6.16, 13.9, and 30.5 mg B/L
Details on test conditions:
Spiked sediments were prepared by addition of boric acid solutions (484 mL of appropriate concentration) to 1500 g formulated sediment and thorough mixing. This resulted in a hydration level of approx. 35%.

Test chambers were 1-L glass jars containing approximately 200 g of formulated sediment (spiked with appropriate amount of test substance) Sediment depth was approximately 2 cm. Dilution water (600 mL) was added to each replicate chamber using a deflector to minimize disturbance of the sediment. Water depth was approx 8 cm, so the water: sediment volume ratio was approx 4:1. Polypropylene emergence traps with nylon mesh were used to monitor adult midge emergence. Chambers were left for 2 d to equilibrate before the test started.

At least 7 replicate chambers were prepared for each treatment level and control. Two were used to provide samples for chemical analysis and 3 chambers were used for biological observations. 20 midge larvae were added to each chamber for the chemical and biological observations. Seven different exposures were tested, a control and six treatments. Larvae were impartially added to cups, then the cups randomly assigned to a replicate chamber.

Overlying water was aerated during the test beginning 24 h after addition of the organisms by gentle bubbling. Midges were fed 2.5 to 3 ml of a commercial flake fish food suspension (2 mg/ml) for the first 10 d, then 6 to 8 ml daily for the remainder of the test. Lighting was 16 h: 8 h light:dark cycle with a 30 m transition period. Light intensity was 653 lux. Temperature was maintained at 20 +/- 2 degrees C by keeping the test chambers in a flowing water bath.

Emergence of adult flies was monitored from day 18. Presence of adults or exuviae in traps was monitored. Adult flies were identified by gender.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
37.7 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: boron added
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
37.7 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
emergence rate
Remarks on result:
other: boron added
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
37.7 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
development rate
Remarks on result:
other: boron added
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
37.7 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: gender ratio
Remarks on result:
other: boron added
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
81 mg/kg sediment dw
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
other: all endpoints
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 37.7 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: all endpoints
Remarks on result:
other: boron added
Details on results:
Percent emergence in the controls was 90% which exceeded the minimum of 70% emergence specified in the OECD 218 guideline for valid results.

All chemical and physical parameters for the 28 day study were within expected ranges with the following exceptions:
Temperature: 18.8 to 20.9 degrees C, except for 15 h period where temperature rose to 25.4 degrees due to water bath heater malfunction.
Dissolved oxygen: 4.7 to 9.0 mg/L. Low oxygen (<60% saturation) occurred in one control and one high exposure replicate on day 21. The exceptions in temperature and dissolved oxygen were judged to have no effect on the test results.

Light intensity: 653 lux (day 0).
Reported statistics and error estimates:
Statistical analysis based on one-way ANOVA followed by Dunnett's test. ECx estimates obtained from probit method and trimmed Spearman-Karber method if poor fit in the probit approach.

Measured Sediment Concentrations and Endpoint Values

Group Nominal Exposure (mg B/kg sediment dw) Sediment Mean Measured Concentration (mg B/kg sediment dw) Sediment Mean Concentration as added Boron (mg B-added/kg) Survival  Emergence  Gender Ratio ** Development rate 
Control 0 0.00 -- 90%  90%  0.7 0.046 
T1  6.5 1.18 1.10* 90%  88%  1.2  0.047 
T2  13 2.83 2.76* 92%  92%  1.4  0.049 
T3  25 6.07 6.00* 88%  88%  1.6  0.048 
T4  50 11.3 11.2* 93%  93%  1.5  0.046 
T5  100 19.4 19.3* 87%  85%  1.3  0.048 
T6  200 37.8 37.7* 88%  88%  0.8  0.046 

* Mean value for group minus mean control boron concentration (i.e., added boron), expressed as mg B-added/kg sediment dw.

**Ratio is males:females

The gender ratio was increased in all treatments above the control (more males than females). However, there was no dose-related pattern; the most highly exposed group (T6) was most similar to controls. In addition, the test with spiked water showed virtually the reversed pattern of gender ratio, again with no evident dose-response pattern.

Validity criteria fulfilled:
yes
Conclusions:
Based on all biological measurements, no significant effects were observed in any treatment. Therefore the NOEC for all endpoints (survival, emergence, development rate, and gender ratio) is stated as the maximum exposure tested. Expressed as the geometric mean concentration in sediment, the NOEC is 37.7 mg B-added/kg sediment dw.

Because no effects were observed, no ECx can be determined.

Analyses of the sediment, pore-water and overlying water at test initiation and termination documented that the boron concentrations decreased in the sediment and pore-water, while increasing in the overlying water. Virtually all added boron could be accounted for (85 to 107%) in the 4 highest treatments and slightly lesser fractions (49 to 63%) in the two smaller treatments.

The geometric mean pore-water and overlying water concentrations of the highest treatment group were 68 and 21 mg B/L respectively. These were associated with no observed effects.
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed and documented study according to GLP and international guidelines
Qualifier:
according to guideline
Guideline:
other: whole sediment chronic toxicity testing of boron with the freshwater fatmucket mussel.
Deviations:
no
Remarks:
based on ASTM E2455-06 (ASTM, 2006), OECD 25 (OECD, 2007), Simon (2005), with use of natural sediment in place of reconstituted sediment
Principles of method if other than guideline:
test methods followed those outlined in the previously approved study protocol
GLP compliance:
yes (incl. QA statement)
Test organisms (species):
other: Lampsilis siliquoidea
Details on test organisms:
- Source: Missouri State University
- Gravid femal L. siliquoidea were collected in Missouri and glochidia obtained were inoculated onto largemouth bass at the MSU propagation facility
- Juvenile L.siliquoidea were reared at MSU in semi-closed systems for approx. 2 months at temperatues maintained between 20 and 25 °C in St Joseph river water.
- Feeding was based on recommendations of the ASTM (2006) guidelines and consisted of a suspension mixture of Nannochloropsis algae and reed mariculture shelfish diet.
Study type:
laboratory study
Test type:
semi-static
Water media type:
freshwater
Type of sediment:
natural sediment
Duration:
21 d
Exposure phase:
total exposure duration
Hardness:
74,4 - 111,2 mg CaCO3/L
Test temperature:
19,1 - 20,6 °C
pH:
6,21 - 7,60
Dissolved oxygen:
5,9 - 9,2 mg/L
Ammonia:
0,02 - 0,46 mg/L as N
Nominal and measured concentrations:
0 (control), 81.6, 143.5, 254.8, 452.4, 806.5 mg B/kg sediment dw
sediment concentrations were maintained by spiing boric acid to the overlying water
Details on test conditions:
TEST SYSTEM
- Control water: recon moderately hard lab water (US EPA, 2002)
- Overlying waters were spiked with boric acid to minimize dissolution and removal of boron from the spiked sediments
- Overlying waters were renewed daily at 20% of the total overlying water volume to ensure adequate water quality

EXPOSURE REGIME
- 10 L.siliquoidea per replicate
- 4 individual replicates

TEST CHARACTERISTCS
- Alkalinity: 20 to 88 mg CaCO3/L
- Photo period: 16hf light / 8hr dark
- Light intensity: 238-490 lux

CHARACTERIZATION OF SEDIMENT
- Lot WBS102910
- no sieving was conducted
- 7,7% clay, 76,9 % silt and 15,4 % sand
- TOC 11,5 %
- Background boron concentration: 7,9 mg B/kg dry sediment
- 12,4 % dry weight

Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
284 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
B
Basis for effect:
mortality
Remarks:
(survival)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
80.6 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
B
Basis for effect:
other: biomass (shell length)
Details on results:
NOEC values are below the IC25 value due to high test precision
Reported statistics and error estimates:
Shapiro-Wilk's and Bartlett's test: normal distribution (p>0,01)
Validity criteria fulfilled:
yes
Conclusions:
All test acceptability criteria specified in the study protocal were met.
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed and documented study according to GLP and international guidelines
Qualifier:
according to guideline
Guideline:
other: whole sediment chronic toxicity testing of boron with the freshwater worm Lumbriculus variegatus
Deviations:
no
Remarks:
based on OECD 225 (OECD, 2007), with use of natural sediment in place of reconstituted sediment
Principles of method if other than guideline:
test methods followed those outlined in the previously approved study protocol
GLP compliance:
yes (incl. QA statement)
Test organisms (species):
Lumbriculus variegatus
Details on test organisms:
- Source: Aquatic Research Organisms in Hampton, NH
- L.variegatus were obtained from cultures maintained at 20 to 23 °C in a system routinely used for culture and maintenance of this organism.
Study type:
laboratory study
Test type:
semi-static
Water media type:
freshwater
Type of sediment:
natural sediment
Duration:
28 d
Exposure phase:
total exposure duration
Hardness:
60,8 - 90,4 mg CaCO3/L
Test temperature:
18,1 - 20,5 °C
pH:
5,88 - 7,51
Dissolved oxygen:
5,9 - 9,2 mg/L
Ammonia:
< 0,1 - 0,87 mg/L as N
Nominal and measured concentrations:
0 (control), 100.8, 210.6, 403.2, 806.4, 1613 mg B/kg sediment dw
sediment concentrations were maintained by spiking boric acid to the overlying water
Details on test conditions:
TEST SYSTEM
- Control water: recon moderately hard lab water (US EPA, 2002)
- Overlying waters were spiked with boric acid to minimize dissolution and removal of boron from the spiked sediments
- Overlying waters were renewed daily at 20% of the total overlying water volume to ensure adequate water quality

EXPOSURE REGIME
- 10 worms per replicate
- 4 individual replicates

TEST CHARACTERISTCS
- Alkalinity: 15 to 30 mg CaCO3/L
- Photo period: 16hr light / 8hr dark
- Light intensity: 190 - 440 lux

CHARACTERIZATION OF SEDIMENT
- no sieving was conducted
- 7,7% clay, 76,9 % silt and 15,4 % sand
- TOC 11,5 %
- Background boron concentration: 7,9 mg B/kg dry sediment
- 12,4 % dry weight

Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
< 100.8 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
B
Basis for effect:
growth rate
Remarks:
(total number of worms)
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
201.6 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
B
Basis for effect:
other: biomass
Details on results:
The NOEC values bracket the IC25 value, indicating concurrence of statistical measures for biomass.
The lowest treatment group (100.8 mg B/kg sediment) resulted in approx a 25% reduction in both biomass and number.
Reported statistics and error estimates:
Shapiro-Wilk's and Bartlett's test: normal distribution (p>0,01)
Validity criteria fulfilled:
yes
Conclusions:
All test acceptability criteria specified in the study protocal were met.

Description of key information

OECD 225, GLP, Boric acid, Lumbriculus variegatus, 28 d, semi-static, 0 (control), 100.8, 210.6, 403.2, 806.4, 1613 mg B/kg sediment dw, NOEC < 100.8 mg B/kg sediment dw

OECD draft document, 1998, GLP, Boric acid, Chironomus riparius, static, 28 d, 18, 32, 56, 100, 180 and 320 mg B/kg dry weight of sediment d.w. 4 replicates were used, NOEC 180 mg B/kg sediment dw

Based on OECD 25, GLP, Boric acid, Lampsilis siliquoidea, semi-static, 21 d, 0 (control), 81.6, 143.5, 254.8, 452.4, 806.5 mg B/kg sediment dw, NOEC 284 mg B/kg sediment dw (mortality)

OECD 218, GLP, Boric acid, Chironomus riparius, static, 28 d, Nominal Sediment Concentrations: 0 (control), 6.5, 13, 25, 50, 100, and 200 mg B/kg dry sediment, NOEC 37.7 mg B/KG sediment dw

OECD 219, GLP, boric acid, Chironomus riparius, static, 28 d, Nominal concentrations definitive test : 0 (control), 2.5, 5.0, 10, 20, 40 and 80 mg B/L, NOEC 20.1 mg B/L (mortality), The EC10 for survival would be 42.7 mg B-added/L and the EC10 for percent emergence would be 40.5 mg B-added/L.

Please refer to read-across statement section 13.

Key value for chemical safety assessment

Additional information