2. Methods

2.1 Sites

Eight cattle access points located in five catchments in the east and south of Ireland were sampled for macroinvertebrates and deposited sediment prior to, and one year following exclusion of cattle from streams via fencing (as a part of the Green Low Carbon Agri-environment Scheme (GLAS)) in October 2016 and October 2017, respectively (Figure 1). Three sites (GLAS Sites 1, 2 and 3) were located along the moderate status, second order Blacklion stream in Co. Carlow. Two sites (GLAS Sites 4 and 5) were located on separate, good-high status tributaries of the Munster Blackwater in Co. Cork. GLAS Site 6 was located on the poor status Commons stream, a tributary of the White river in Co. Louth. GLAS Site 7 was located on a good status, first order tributary of the Big river in Co. Louth. GLAS Site 8 was on a moderate status tributary of the Milltown Lake catchment in Co. Monaghan. Geology was variable (see supplementary information) and across sites while catchment land use was principally grassland. Stream substrates at GLAS Sites 1, 2, 4 and 7 were predominately gravel whereas coarse substrates, mainly cobble, dominated at all other sites. Stream widths ranged from 0.72m to 3.60 m (supplementary information).
The study on the long-term efficacy of fencing was carried out in the Milltown Lake catchment of Co. Monaghan (Figure 1). Here 13 sites were sampled in October 2008 (see Wynne and Linnane, 2008). Seven sites were located in a sub-catchment fed from the Carnagh lake in Tievenamara (TV), that was entirely fenced off from cattle following sampling in 2008, as part of remediation works for a group water scheme. Six sites were located in a control catchment in the Tullycaghney (TH) that was not fenced as a part of a concerted effort (although some ad-hoc, localised fencing has taken place since sampling in 2008). All sites were re-sampled in October 2017, nine years post fencing.

2.2 Macroinvertebrate sampling

For the short-term fencing study, twelve macroinvertebrate samples were collected at each site prior to fencing (in October 2016) and one-year post fencing (in October 2017) using a standard 0.9m2Surber sampler. Sampling points were located in riffle geomorphic units immediately upstream (x 6) and downstream (x 6) of cattle access points, hereafter referred to as control and pressure points, respectively. A total of 96 samples (six Surbers per sampling point) were collected in each year. The majority of insect taxa were identified to species level; Coleoptera and Diptera were identified to family level and oligochaetes were left at order level.
For the long-term fencing study, samples were collected (in 2008 and 2019) by 2-minute kick sampling from riffle habitats at each sampling point. To enable comparisons with the pre-fencing study (Wynne and Linnane, 2008) the sampling methods of Wynne and Linnane were replicated as closely as possible and the macroinvertebrates were identified to family level.

2.3 Sediment sampling

Deposited sediment sampling was undertaken as part of the short-term study only. The samples were taken from riffle habitats at control and pressure points at the same time as macroinvertebrate sampling, using the ‘Quorer’ resuspendable sediment sampling technique (Lambert and Walling, 1988; Conroy et al., 2016b). Six samples were collected at each sampling point resulting in a total of 96 deposited sediment samples from each sampling period.
The deposited sediment samples were filtered using pre-ashed Whatman glass Microfibre GF/C filters following standard methods for suspended sediment as set out by the American Public Health Association (APHA, 1995). They dried at 103⁰C to a constant weight and weighed to give sediment mass (g). Organic matter mass was determined by weights of filter papers upon loss on ignition of samples dried at 550⁰C to a constant weight (APHA, 1995). Estimated resuspended sediment was calculated as mg l-1 and converted to g m-2 using the water volume within the stilling well (Conroy et al., 2016b). Percent organic matter (%OM) was calculated as a percentage of overall sediment mass.

2.4 Habitat assessment

Habitat assessment was carried out as part of the short-term study prior to fencing (2016) and following one year of exclusion (2017). It consisted of both quantitative measures of stream bank parameters and an assessment of qualitative parameters, carried out at the reach scale. A total of 13 qualitative sub-indices were calculated and were summed to produce a Total Habitat Index (THI) and a Riparian Habitat Index (RHI) (see O’Sullivan et al., 2019a). Each habitat assessment sub-index was scored on an ordinal scale from 0-20 or 0-10 where stream banks were assessed individually, with higher scores equating to better habitat condition.

2.5 Data analysis

Short-term study
A before, after, control and impact (BACI) study design was used to test whether short-term exclusion of cattle via fencing affected levels of deposited stream sediment metrics and macroinvertebrate community structure. The analyses of macroinvertebrate data tested the hypothesis that differences in community structure between control and pressure points prior to fencing would be greater than those differences following fencing and one year of cattle exclusion. This hypothesis was tested in Primer using the PERMDISP function (Anderson, 2006).
Here, Site (levels: 1-8) and Fencing (levels: Pre and Post) factors were combined to create the grouping factor for the PERMDISP procedure. Thus, samples from control and pressure points were combined for each site and the homogeneity of dispersions between samples (taking in both control and pressure samples) prior to and following fencing were tested via permutations, as is the protocol with the PERMDISP procedure. The hypothesis, thus states that homogeneity of dispersions will increase following fencing, or conversely, the within group variation will be reduced following fencing (Figure 2).
Pairwise comparisons of PERMDISP results and mean z scores were assessed to identify any convergence or divergence of samples between pre-fencing and post-fencing time periods at each site.
Potential differences in the multivariate locations (centroids) of macroinvertebrate samples (community structure) based on control v pressure differences before and after fencing were tested using PERMANOVA. In these analyses Fencing and Treatment were combined to create a fixed factor with four levels (Pre-fencing control, pre-fencing pressure, post-fencing control and post-fencing pressure).Site was a random factor with eight levels (GLAS Sites 1, 2, 3, 4, 5, 6, 7 and 8).
SIMPER analysis was used to identify taxa that contributed to dissimilarities in community structure between four combinations ofFencing/Treatment levels:
  1. pre-fencing and post-fencing control points
  2. pre-fencing and post-fencing pressure points
  3. pre-fencing control and pre-fencing pressure points
  4. post-fencing control and post-fencing pressure points.
These SIMPER comparisons were made using data from across all eight sites to identify taxa that demonstrated consistent changes to exclusion.
Univariate metrics (Ephemeroptera Plecoptera Trichoptera (EPT) abundance, % EPT, richness indices, functional feeding group (FFG) metrics and fine sediment sensitivity rating (FSSR) metrics) were also analysed using this study design. Pairwise tests for pairs of theFencing/Treatment term within levels of the Site term were carried out to investigate any interaction effects in relation to univariate metrics.
Mean deposited sediment values were calculated for each Treatment level (control and pressure) at each site for the pre-fencing and post-fencing periods. PERMANOVA analysis was carried out using a two-factor design with Fencing and Treatment as two fixed factors, each with two levels (pre-fencing and post-fencing, and control and pressure, respectively).
With respect to habitat, qualitative habitat assessment scores were analysed as univariate data using a two factor PERMANOVA (based on Euclidean distance) with Fencing and Treatment as fixed factors. Habitat scores were square root transformed prior to analysis.

2.6 Long-term study

A blocked study design was used to assess for effects of long-term fencing on macroinvertebrate community structure, using a three factor PERMANOVA. The three factors were Fencing and Catchment(both fixed with two levels; pre-fencing and post-fencing and fenced (TV) and control (TH) respectively) and Site nested inCatchment .
Blocked study designs as carried out in Primer v.6 are akin to repeated measure PERMANOVAs and proceed with the exclusion of the ‘highest level’ interaction term (Site(Catchment) x Fencing in this case) from the model (Anderson et al., 2008). Blocking of un-replicated samples leads to greater power of detection (Fisher, 1935).